Ca_V channels cluster at the active zone

Physiological experiments have established that the voltage-gated calcium (Ca_V) channels in rod and cone photoreceptors are high voltage-activated L-type channels (Bader et al., Reference Bader, Bertrand and Schwartz1982; Corey et al., Reference Corey, Dubinsky and Schwartz1984; Barnes & Hille, Reference Barnes and Hille1989; Lasater & Witkovsky, Reference Lasater and Witkovsky1991; Wilkinson & Barnes, Reference Wilkinson and Barnes1996). There is no evidence for non-L-type Ca_V channels in photoreceptors. Freeze-fracture electron micrographs of photoreceptor terminals show a random array of hexagonal-shaped particles at the apex of the synaptic ridge that are thought to be Ca_V channels (Raviola & Gilula, Reference Raviola and Gilula1975; Schaeffer et al., Reference Schaeffer, Raviola and Heuser1982). Terminals of rods and cones in the outer plexiform layer (OPL) label with antibodies to pore-forming Ca_V1.2 (α_1C), Ca_V1.3 (α_1D), and Ca_V1.4 (α_1F) subunit isoforms (Nachman-Clewner et al., Reference Nachman-Clewner, St Jules and Townes-Anderson1999; Firth et al., Reference Firth, Morgan, Boelen and Morgans2001; Henderson et al., Reference Henderson, Doerr, Gottesman and Miller2001; Morgans, Reference Morgans2001; Morgans et al., Reference Morgans, Gaughwin and Maleszka2001, Reference Morgans, Bayley, Oesch, Ren, Akileswaran and Taylor2005; Wu et al., Reference Wu, Marmorstein, Striessnig and Peachey2003; Mansergh et al., Reference Mansergh, Orton, Vessey, Lalonde, Stell, Tremblay, Barnes, Rancourt and Bech-Hansen2005; tom Dieck et al., Reference tom Dieck, Altrock, Kessels, Qualmann, Regus, Brauner, Fejtová, Bracko, Gundelfinger and Brandstätter2005; Xiao et al., Reference Xiao, Chen and Steele2007; Specht et al., Reference Specht, Wu, Turner, Dearden, Koentgen, Wolfrum, Maw, Brandstätter and tom Dieck2009; Kersten et al., Reference Kersten, van Wijk, van Reeuwijk, van der Zwaag, Märker, Peters, Katsanis, Wolfrum, Keunen, Roepman and Kremer2010; Mercer et al., Reference Mercer, Chen and Thoreson2011a,Reference Mercer, Rabl, Riccardi, Brecha, Stella and Thoresonb). High levels of Ca_V1.3 and Ca_V1.4 messenger RNA (mRNA) are present in retina (Bech-Hansen et al., Reference Bech-Hansen, Naylor, Maybaum, Pearce, Koop, Fishman, Mets, Musarella and Boycott1998; Strom et al., Reference Strom, Nyakatura, Apfelstedt-Sylla, Hellebrand, Lorenz, Weber, Wutz, Gutwillinger, Rüther, Drescher, Sauer, Zrenner, Meitinger, Rosenthal and Meindl1998; Xiao et al., Reference Xiao, Chen and Steele2007) but only low levels of Ca_V1.2 mRNA (Kamphuis & Hendriksen, Reference Kamphuis and Hendriksen1998), suggesting that the labeling of photoreceptors by Ca_V1.2 antibodies may be nonspecific. Mutations in Ca_V1.4 greatly diminish the electroretinogram (ERG) b-wave in knockout mice, produce malformed ribbons, and cause congenital stationary night blindness (CSNB) in humans. Combined with immunohistochemical evidence, these findings suggest that Ca_V1.4 is the principle Ca_V channel subtype in rods (Bech-Hansen et al., Reference Bech-Hansen, Naylor, Maybaum, Pearce, Koop, Fishman, Mets, Musarella and Boycott1998; Strom et al., Reference Strom, Nyakatura, Apfelstedt-Sylla, Hellebrand, Lorenz, Weber, Wutz, Gutwillinger, Rüther, Drescher, Sauer, Zrenner, Meitinger, Rosenthal and Meindl1998; Mansergh et al., Reference Mansergh, Orton, Vessey, Lalonde, Stell, Tremblay, Barnes, Rancourt and Bech-Hansen2005; Chang et al., Reference Chang, Heckenlively, Bayley, Brecha, Davisson, Hawes, Hirano, Hurd, Ikeda, Johnson, McCall, Morgans, Nusinowitz, Peachey, Rice, Vessey and Gregg2006; Striessnig et al., Reference Striessnig, Bolz and Koschak2010).

The contribution of Ca_V1.3 at photoreceptor synapses is less clear. Immunoelectron micrographs show labeling for antibodies to Ca_V1.3 at the base of ribbons in mouse retina (Kersten et al., Reference Kersten, van Wijk, van Reeuwijk, van der Zwaag, Märker, Peters, Katsanis, Wolfrum, Keunen, Roepman and Kremer2010). However, Ca_V1.3 mutations in mouse retina do not significantly diminish the b-wave (Wu et al., Reference Wu, Marmorstein, Striessnig and Peachey2007), and b-waves of Ca_V1.3/Ca_V1.4 double mutants do not appear to differ from those of Ca_V1.4 single mutants (McCall & Gregg, Reference McCall and Gregg2008). It may be that Ca_V1.3 channels are present only in a subpopulation of cones. For example, Ca_V1.3 antibodies label M cones but not S cones in the tree shrew retina (Morgans, Reference Morgans1999). Subtype differences among cones also occur in salamander retina where protein kinase A (PKA) inhibits Ca²⁺ currents in rods and short wavelength-sensitive small single cones but enhances Ca²⁺ currents of long wavelength-sensitive large single cones (Stella & Thoreson, Reference Stella and Thoreson2000). PKA activation also enhances currents from heterologously expressed Ca_V1.3 channels (Qu et al., Reference Qu, Baroudi, Yue, El-Sherif and Boutjdir2005; Liang & Tavalin, Reference Liang and Tavalin2007). The pharmacological properties of Ca²⁺ currents, including partial block by omega-conotoxin, are consistent with the presence of Ca_V1.3 channels in salamander cones (Wilkinson & Barnes, Reference Wilkinson and Barnes1996). Antibodies to α₂δ₄ subunits label photoreceptor terminals (Mercer et al., Reference Mercer, Chen and Thoreson2011a), and mutations in β₂ or α₂δ₄ subunits cause diminished ERG b-waves and CSNB, suggesting that these are the principle Ca_V channel accessory subunits in photoreceptors (Ball et al., Reference Ball, Powers, Shin, Morgans, Peachey and Gregg2002; Wycisk et al., Reference Wycisk, Budde, Feil, Skosyrski, Buzzi, Neidhardt, Glaus, Nürnberg, Ruether and Berger2006a,Reference Wycisk, Zeitz, Feil, Wittmer, Forster, Neidhardt, Wissinger, Zrenner, Wilke, Kohl and Bergerb).

Excision of the synaptic terminal diminishes rod calcium currents by 95%, indicating that most of the Ca_V channels are located in the terminal (Xu & Slaughter, Reference Xu and Slaughter2005). As illustrated in Fig. 1, sites of intraterminal Ca²⁺ influx also colocalize with synaptic ribbons in rod photoreceptor terminals. In this example, changes in intracellular Ca²⁺ levels were visualized in a salamander rod using the Ca²⁺-sensitive dye, Oregon Green BAPTA-6F, (Invitrogen, Carlsbad, CA) (K _D = 3 μM Ca²⁺), and the ribbon was labeled with a rhodamine-conjugated consensus peptide that binds selectively to the ribbon protein, ribeye (Zenisek et al., Reference Zenisek, Horst, Merrifield, Sterling and Matthews2004). Both dyes were introduced into the rod through a patch-clamp pipette. Sites of Ca²⁺ influx and ribbons are also colocalized in cones (Choi et al., Reference Choi, Jackman, Thoreson and Kramer2008). Consistent with close proximity between Ca_V channels and the ribbon, immunohistochemical studies provide evidence for clusters of Ca_V channels at the base of the ribbon (Nachman-Clewner et al., Reference Nachman-Clewner, St Jules and Townes-Anderson1999; Morgans, Reference Morgans2001; Morgans et al., Reference Morgans, Bayley, Oesch, Ren, Akileswaran and Taylor2005; Specht et al., Reference Specht, Wu, Turner, Dearden, Koentgen, Wolfrum, Maw, Brandstätter and tom Dieck2009; Mercer et al., Reference Mercer, Rabl, Riccardi, Brecha, Stella and Thoreson2011b). Furthermore, individual Ca_V channels labeled with quantum dots conjugated to α₂δ₄ antibodies colocalize with fluorescently labeled ribeye consensus peptides (Mercer et al., Reference Mercer, Chen and Thoreson2011a), and immunoelectron micrographs show Ca_V channels along the synaptic ridge at the base of the ribbon (tom Dieck et al., Reference tom Dieck, Altrock, Kessels, Qualmann, Regus, Brauner, Fejtová, Bracko, Gundelfinger and Brandstätter2005; Kersten et al., Reference Kersten, van Wijk, van Reeuwijk, van der Zwaag, Märker, Peters, Katsanis, Wolfrum, Keunen, Roepman and Kremer2010).

Fig. 1.

Colocalization of Ca²⁺ channels to ribbon release sites. A voltage-clamped rod from a salamander retinal slice preparation was loaded with Oregon Green BAPTA-6F (OGB-6F) (200 μM) to visualize intracellular Ca²⁺ changes and a rhodamine-conjugated ribeye-binding peptide (50 μM; Zenisek et al., Reference Zenisek, Horst, Merrifield, Sterling and Matthews2004) to visualize the ribbon. Both were introduced into the rod through a patch pipette. To activate L-type Ca²⁺ channels, the voltage-clamped rod was depolarized from −70 to −10 mV for 100 ms. Panel (A) shows a single confocal section (55 ms exposure) illustrating OGB-6F fluorescence in the rod prior to the test step. Panel (B) shows OGB-6F fluorescence in the same confocal section during the depolarizing test step. The difference image (test step minus control) in panel (C) shows localized hot spots of Ca²⁺ increase at the base of the synaptic terminal (arrows). Panel (D) shows synaptic ribbons labeled with rhodamine-conjugated ribeye-binding peptide in the same rod. Note that locations of the ribbons match the sites of Ca²⁺ influx in the two terminals of the rod. OGB-6F was imaged with 488 nm excitation and 525 nm emission filters, and rhodamine was visualized using 568 nm excitation and 607 nm emission filters. Scale bar = 10 μm.

Ca²⁺-dependent vesicle release and replenishment at photoreceptor synapses

At most synapses, Ca²⁺ needs to attain high micromolar levels to stimulate release, and therefore, exocytosis is typically restricted to release sites within nanodomains of high Ca²⁺ very close to Ca_V channels. A Ca²⁺ nanodomain is defined as the 10–100 nm range in the immediate vicinity of a Ca²⁺ channel where Ca²⁺ is not in equilibrium with fast buffers (Naraghi & Neher, Reference Naraghi and Neher1997). Examples of synapses where release involves Ca²⁺ nanodomains include the squid giant synapse (Adler et al., Reference Adler, Augustine, Duffy and Charlton1991), mammalian rod bipolar cell (Jarsky et al., Reference Jarsky, Tian and Singer2010), GABAergic basket cells in the hippocampus (Bucurenciu et al., Reference Bucurenciu, Kulik, Schwaller, Frotscher and Jonas2008), and mature calyx of Held (Fedchyshyn & Wang, Reference Fedchyshyn and Wang2005; Weber et al., Reference Weber, Wong, Tufford, Schlichter, Matveev and Stanley2010). The release mechanism in rods and cones is much more sensitive to Ca²⁺ than at other synapses, and exocytosis can thus be triggered by submicromolar Ca²⁺ levels (Rieke & Schwartz, Reference Rieke and Schwartz1996; Thoreson et al., Reference Thoreson, Rabl, Townes-Anderson and Heidelberger2004; Sheng et al., Reference Sheng, Choi, Dharia, Li, Sterling and Kramer2007; Duncan et al., Reference Duncan, Rabl, Gemp, Heidelberger and Thoreson2010). The high Ca²⁺ affinity of the release mechanism at photoreceptor synapses suggests that release might involve spatially averaged Ca²⁺ levels in the terminal and not the high Ca²⁺ levels found in nanodomains. However, experiments comparing effects of the diffusible synthetic Ca²⁺ chelators ethylene glycol tetraacetic acid (EGTA) and 1,2-bis (o-aminophenoxy) ethane,N,N,N',N'-tetraacetic acid (BAPTA) on release indicate that release sites are within 50–100 nm of Ca²⁺ channels in amphibian cones (Mercer et al., Reference Mercer, Rabl, Riccardi, Brecha, Stella and Thoreson2011b). This was shown by the finding that when 0.5 mM EGTA, 5 mM EGTA, or 5 mM BAPTA were introduced into cones, only 5 mM BAPTA reduced the amplitude of glutamatergic post-synaptic currents in second-order neurons (Mercer et al., Reference Mercer, Rabl, Riccardi, Brecha, Stella and Thoreson2011b). Comparisons of the calcium current to the size of the readily releasable pool of synaptic vesicles suggest that only a few Ca²⁺ channel openings accompany fusion of each vesicle during the first few milliseconds of release from cones (Bartoletti et al., Reference Bartoletti, Babai and Thoreson2010). Thus, when cones are depolarized by a light decrement, the release of vesicles is triggered by the emergence of nanodomains of [Ca²⁺]_i beneath open Ca_V channels close to ribbon release sites, providing a tight coupling between channel opening and vesicle fusion (Mercer et al., Reference Mercer, Rabl, Riccardi, Brecha, Stella and Thoreson2011b).

In darkness, cones appear to switch from a tight coupling between Ca_V channel opening and vesicle fusion to a mode of release whereby the rate of sustained release is governed by the rate of replenishment. At the dark potential, cones are depolarized to a membrane potential of approximately −40 mV, allowing the continued influx of Ca²⁺, which in turn causes a depletion of the readily releasable pool of vesicles at the base of the ribbon (Jackman et al., Reference Jackman, Choi, Thoreson, Rabl, Bartoletti and Kramer2009; Bartoletti et al., Reference Bartoletti, Babai and Thoreson2010). Under these conditions, the rate of synaptic release is limited not by the rate of individual channel openings but by the rate at which release-ready vesicles can be replenished (Jackman et al., Reference Jackman, Choi, Thoreson, Rabl, Bartoletti and Kramer2009). The replenishment process is Ca²⁺ dependent (Babai et al., Reference Babai, Bartoletti and Thoreson2010) and so, even when the releasable pool is depleted in darkness, release is nonetheless regulated by Ca²⁺ influx through Ca_V channels. However, the Ca²⁺-dependent sites involved in replenishment are >200 nm from Ca_V channels, further than the distance between release sites and channels (Babai et al., Reference Babai, Bartoletti and Thoreson2010). Because these Ca²⁺-dependent sites of replenishment are not located within Ca²⁺ nanodomains beneath individual channels, replenishment is regulated by the spatially averaged levels of Ca²⁺ entering through multiple channels. In bright light, random channel openings are rare, and individual channel openings are closely synchronized with decrements in light intensity. Under these conditions, close coupling between channel openings and release events can provide an accurate read out of stimulus-dependent changes in membrane potential. When cones are depolarized in darkness, many Ca²⁺ channel openings occur at random intervals. Controlling release under these conditions by regulating the rate of replenishment offers the benefit of reducing noise that can be introduced by stochastic channel openings.

Evidence that the Ca²⁺-dependent replenishment mechanism involves sites >200 nm from Ca_V channel suggests that proteins along the ribbon may participate in this process. At the calyx of Held, calmodulin plays a key role in the Ca²⁺-dependent acceleration of replenishment (Sakaba & Neher, Reference Sakaba and Neher2001). One target of calmodulin is the GTP-binding protein, Rab3a (Park et al., Reference Park, Kim, Lee, Kim, Seo and Kim2002), and Rab3a has been shown to play a role in replenishment (Leenders et al., Reference Leenders, Lopes da Silva, Ghijsen and Verhage2001). Rab3a interacts with RIM2 proteins along the ribbon and has been proposed to tether vesicles to the ribbon (Fukuda, Reference Fukuda2003; Dulubova et al., Reference Dulubova, Lou, Lu, Huryeva, Alam, Schneggenburger, Südhof and Rizo2005; tom Dieck et al., Reference tom Dieck, Altrock, Kessels, Qualmann, Regus, Brauner, Fejtová, Bracko, Gundelfinger and Brandstätter2005; Deguchi-Tawarada et al., Reference Deguchi-Tawarada, Inoue, Takao-Rikitsu, Inoue, Kitajima, Ohtsuka and Takai2006; Uthaiah & Hudspeth, Reference Uthaiah and Hudspeth2010). Along with calmodulin, Rab3a is an appealing candidate for regulating replenishment at the cone ribbon.

Synaptic Ca²⁺ buffering and extrusion

Ca²⁺ influx can induce vesicle release and regulate replenishment, but rising [Ca²⁺]_i levels are balanced by Ca²⁺ extrusion and buffering mechanisms. One of the first studies of synaptic Ca²⁺ extrusion in photoreceptors was performed by Morgans et al. (Reference Morgans, El Far, Berntson, Wässle and Taylor1998) to determine whether the Na⁺/Ca²⁺ exchanger or the plasma membrane Ca²⁺ ATPase (PMCA) tempered increases in [Ca²⁺]_i following a depolarizing stimulus. PMCA is considered critical for Ca²⁺ handling at other synapses (Juhaszova et al., Reference Juhaszova, Church, Blaustein and Stanley2000; Wanaverbecq et al., Reference Wanaverbecq, Marsh, Al-Qatari and Brown2003; Gover et al., Reference Gover, Moreira, Kao and Weinreich2007), but the Na⁺/Ca²⁺ exchanger regulates Ca²⁺ levels in the outer segments of rod photoreceptors (Thoreson et al., Reference Thoreson, Nitzan and Miller1997; Lagnado et al., Reference Lagnado, Cervetto and McNaughton1998; Krizaj & Copenhagen, Reference Krizaj and Copenhagen2002). Morgans et al. (Reference Morgans, El Far, Berntson, Wässle and Taylor1998) demonstrated that the PMCA handled Ca²⁺ extrusion in terminals of cone photoreceptors from the tree shrew, and that photoreceptors in the tree shrew, mouse, goldfish, and rat express a PMCA protein using a pan-PMCA channel antibody. Labeling with PMCA antibodies is found just above antibodies to Ca_V1.3 channels, colocalized with presynaptic postsynaptic density-95 (PSD-95) complexes and the Crumbs complex protein membrane palmitoylated protein 4 (MPP4) (Yang et al., Reference Yang, Pawlyk, Wen, Adamian, Soloviev, Michaud, Zhao, Sandberg, Makino and Li2007; Aartsen et al., Reference Aartsen, Arsanto, Chauvin, Vos, Versteeg, Cardozo, Bivic and Wijnholds2009). Therefore, it seems reasonable to suggest that PMCA transporters lie some distance from synaptic release sites to avoid quenching brief Ca²⁺ signals in the active zone during dim light conditions but capable of limiting global increases in [Ca²⁺]_i throughout the terminal. Similar PMCA expression patterns are found in the retinas of mice (Krizaj et al., Reference Krizaj, Demarco, Johnson, Strehler and Copenhagen2002; Cia et al., Reference Cia, Bordais, Varela, Forster, Sahel, Rendon and Picaud2005; Johnson et al., Reference Johnson, Perkins, Giddabasappa, Chaney, Xiao, White, Brown, Waggoner, Ellisman and Fox2007), tiger salamanders (Krizaj et al., Reference Krizaj, Lai and Copenhagen2003, Reference Krizaj, Liu and Copenhagen2004), and chickens (Tolosa de Talamoni et al., Reference Tolosa de Talamoni, Pérez, Riis, Smith, Norman and Wasserman2002). However, using a C57Bl/6J mouse model, Johnson et al. (Reference Johnson, Perkins, Giddabasappa, Chaney, Xiao, White, Brown, Waggoner, Ellisman and Fox2007) found that PMCA is critical for Ca²⁺ extrusion in rod terminals, whereas the Na⁺/Ca²⁺ exchanger is more important for maintaining Ca²⁺ homeostasis in cone terminals. This corroborates the finding that PMCA2 knockout mice exhibit deficits only in rod-mediated ERG responses (Duncan et al., Reference Duncan, Yang, Doan, Silverstein, Murphy, Nune, Liu, Copenhagen, Tempel, Rieke and Krizaj2006) and calls into question the idea that PMCAs are solely responsible for Ca²⁺ homeostasis in the synaptic terminals of all photoreceptor cells.

Although Ca²⁺ extrusion can modulate Ca²⁺ homeostasis at photororeceptor synapses, endogenous buffering systems are also important (Thoreson, Reference Thoreson2007). Extrusion of Ca²⁺ through PMCA or the Na⁺/Ca²⁺ exchanger is slow compared to the kinetics of Ca²⁺ buffering (Klingauf & Neher, Reference Klingauf and Neher1997; Thoreson et al., Reference Thoreson, Nitzan and Miller1997; Krizaj et al., Reference Krizaj, Liu and Copenhagen2004; Mercer et al., Reference Mercer, Rabl, Riccardi, Brecha, Stella and Thoreson2011b). Local calcium buffering shapes diffusion of Ca²⁺ influx at the active zone (Klingauf & Neher, Reference Klingauf and Neher1997) and thereby shapes the kinetics of neurotransmission. Endogenous neuronal buffers include calbindin, calretinin, and parvalbumin (Bennis et al., Reference Bennis, Versaux-Botteri, Repérant and Armengol2005; Rusakov, Reference Rusakov2006). Although there is significant species-to-species variability, cone photoreceptors in most species contain calbindin, a fast low mobility buffer, while calretinin and parvalbumin are only observed in the photoreceptors of a handful of species.

The calcium-binding proteins CaBP4 (Haeseleer et al., Reference Haeseleer, Imanishi, Maeda, Possin, Maeda, Lee, Rieke and Palczewski2004; Lee et al., Reference Lee, Jimenez, Cui and Haeseleer2007) and calmodulin (Miller, Reference Miller1991; Griessmeier et al., Reference Griessmeier, Cuny, Rötzer, Griesbeck, Harz, Biel and Wahl-Schott2009) are also present at photoreceptor synapses and can buffer relatively high (>1 μM) increases in [Ca²⁺]_i. CaBP4 shifts the voltage activation range of Ca_V1.4 towards more negative potentials, allowing channels to be effectively activated by physiological voltage levels below −40 mV (Haeseleer et al., Reference Haeseleer, Imanishi, Maeda, Possin, Maeda, Lee, Rieke and Palczewski2004). CaBP4 weakly inhibits calcium-dependent inactivation of Ca_V1.3 (Cui et al., Reference Cui, Meyer, Calin-Jageman, Neef, Haeseleer, Moser and Lee2007) and is a phosphorylation target of intracellular kinase pathways (Lee et al., Reference Lee, Jimenez, Cui and Haeseleer2007).

The synaptic ribbon

A striking feature of many sensory synapses is the presence of a large electron-dense structure known as the ribbon. Although its function in vesicle release is not yet fully understood, it is believed that the ribbon assists sensory neurons in maintaining graded tonic neurotransmission. Ribbons were initially proposed to function as conveyor belts that help transport vesicles to the active zone for exocytosis (Bunt, Reference Bunt1971). However, cytosolic ATP is not needed to release vesicles that have already been primed and attached to the ribbon, indicating that vesicle movement along the ribbon does not involve a molecular motor requiring ATP hydrolysis (Heidelberger, Reference Heidelberger1998). In fact, rather than accelerating vesicle replenishment at the base of the ribbon, the observation that vesicles are depleted from the base of the ribbon in darkness suggests that ribbons actually slow vesicle delivery (Jackman et al., Reference Jackman, Choi, Thoreson, Rabl, Bartoletti and Kramer2009). Other proposed functions include the possibility that ribbons may prime vesicles for release, act like a flytrap to capture and deliver vesicles to the active zone, assist with endocytosis of previously released vesicles, or coordinate multivesicular release (Prescott & Zenisek, Reference Prescott and Zenisek2005).

Ribbons are found in bipolar and photoreceptor cells of the retina, pineal photoreceptors, hair cells, and electroreceptors. Ultrastructural studies show differences in the shapes of ribbons in different cell types. For example, ribbons of rod and cone photoreceptors and bipolar cells are typically more plate-like, whereas ribbons in hair cells are often spherical in shape (tom Dieck & Brandstätter, Reference tom Dieck and Brandstätter2006). Synaptic ribbons in vertebrate photoreceptors are first expressed as similar precursor spheres (Regus-Leidig et al., Reference Regus-Leidig, tom Dieck, Specht, Meyer and Brandstätter2009). Spherical ribbons are also found in photoreceptors from the hagfish retina suggesting that this may represent the primordial shape of the ribbon (Holmberg & Ohman, Reference Holmberg and Ohman1976). The main protein in the ribbon, ribeye, does not appear to be present in invertebrates, but photoreceptors from Drosophila melanogaster have T-bar structures that are sometimes compared to ribbons (Fouquet et al., Reference Fouquet, Owald, Wichmann, Mertel, Depner, Dyba, Hallermann, Kittel, Eimer and Sigrist2009; Hamanaka & Meinertzhagen, Reference Hamanaka and Meinertzhagen2010). Conventional vertebrate synapses also sometimes exhibit structures that resemble ribbons, such as spherical structures at the frog neuromuscular junction and punctate electron-dense projections at synapses between human hippocampal neurons (Zhai & Bellen, Reference Zhai and Bellen2004).

Ribbons at synapses of vertebrate rod and cone photoreceptors are typically 30–40 nm wide and project 200–400 nm vertically into the cytoplasm (reviewed by Heidelberger et al., Reference Heidelberger, Thoreson and Witkovsky2005; Sterling & Matthews, Reference Sterling and Matthews2005; Schmitz, Reference Schmitz2009). Cone ribbons have a base extending 200–700 nm along the membrane, whereas rod ribbons are 800–1500 nm long. Bipolar cell ribbons are normally smaller than photoreceptor ribbons (Sterling & Matthews, Reference Sterling and Matthews2005). Terminals of mammalian rods typically contain one ribbon, whereas amphibian rods have multiple ribbons (Carter-Dawson & LaVail, Reference Carter-Dawson and LaVail1979; Townes-Anderson et al., Reference Townes-Anderson, MacLeish and Raviola1985; Migdale et al., Reference Migdale, Herr, Klug, Ahmad, Linberg, Sterling and Schein2003; Zampighi et al., Reference Zampighi, Schietroma, Zampighi, Woodruff, Wright and Brecha2011), but both mammalian and nonmammalian cones have larger terminals with a dozen or more ribbons. Vesicles tethered along the bottom one or two rows of the ribbon contact the plasma membrane and appear to form a readily releasable pool of vesicles that can fuse rapidly following an increase in [Ca²⁺]_i (Sterling & Matthews, Reference Sterling and Matthews2005; Bartoletti et al., Reference Bartoletti, Babai and Thoreson2010). A single ribbon at a rod synapse tethers ∼25 vesicles to the plasma membrane at the base of the ribbon with a total tethered pool of ∼700 vesicles (Thoreson et al., Reference Thoreson, Rabl, Townes-Anderson and Heidelberger2004; Heidelberger et al., Reference Heidelberger, Thoreson and Witkovsky2005), whereas cone ribbons have a readily releasable pool of ∼20 vesicles and a total tethered pool of ∼110 vesicles (Innocenti & Heidelberger, Reference Innocenti and Heidelberger2008; Bartoletti et al., Reference Bartoletti, Babai and Thoreson2010).

Ribeye

Technological advances over the past 20 years have allowed scientists to characterize many of the key structural and functional domains of photoreceptor ribbons. The primary protein constituent of the ribbon is ribeye, which was first identified, characterized, and cloned by Schmitz et al. (Reference Schmitz, Königstorfer and Südhof2000). Ribeye is composed of a novel proline-rich A domain and a carboxyterminal B domain that is almost identical in sequence to the nuclear repressor, CtBP2. Both protein products are encoded by a single gene, but ribeye is expressed only at ribbon synapses (Wan et al., Reference Wan, Almers and Chen2005; Magupalli et al., Reference Magupalli, Schwarz, Alpadi, Natarajan, Seigel and Schmitz2008). Synaptic ribbons have been visualized by fluorescent and electron microscopy using antibodies to ribeye (Schmitz et al., Reference Schmitz, Königstorfer and Südhof2000), antibodies to CtBP2 (tom Dieck et al., Reference tom Dieck, Altrock, Kessels, Qualmann, Regus, Brauner, Fejtová, Bracko, Gundelfinger and Brandstätter2005; Uthaiah & Hudspeth, Reference Uthaiah and Hudspeth2010), and fluorescently labeled ribeye consensus peptides (Zenisek et al., Reference Zenisek, Horst, Merrifield, Sterling and Matthews2004; Choi et al., Reference Choi, Jackman, Thoreson and Kramer2008; LoGiudice et al., Reference LoGiudice, Sterling and Matthews2008; Zenisek, Reference Zenisek2008).

The A domain of ribeye constitutes most of the internal scaffold of the ribbon structure, while the B domain faces into the cytoplasm where it can interact with other ribbon-related proteins (Schmitz et al., Reference Schmitz, Königstorfer and Südhof2000; Alpadi et al., Reference Alpadi, Magupalli, Käppel, Köblitz, Schwarz, Seigel, Sung and Schmitz2008; Magupalli et al., Reference Magupalli, Schwarz, Alpadi, Natarajan, Seigel and Schmitz2008). The scaffold of the ribbon is constructed from homotypic and heterotypic interactions between A and B domains of adjacent ribeye molecules. Heterotypic interactions between A and B domains are inhibited by nicotinamide adenine dinucleotide [NAD(H)] (Magupalli et al., Reference Magupalli, Schwarz, Alpadi, Natarajan, Seigel and Schmitz2008).

Ribbon-associated proteins

Although the backbone of a ribbon is constructed from interlinking ribeye peptides, a number of accessory cytomatrix proteins at the synapse appear to help organize vesicle trafficking near the ribbon (Zanazzi & Matthews, Reference Zanazzi and Matthews2009). Directly beneath the ribbon in photoreceptor cells, but not most other ribbon synapses, is a trough-like electron-dense structure called the arciform density (Dowling & Boycott, Reference Dowling and Boycott1966; Gray & Pease, Reference Gray and Pease1971; Lasansky, Reference Lasansky1973; Raviola & Gilula, Reference Raviola and Gilula1975; Raviola & Raviola, Reference Raviola and Raviola1982). The arciform density may help to anchor the ribbon to the plasma membrane and perhaps shape the diffusion of Ca²⁺ entering through Ca²⁺ channels beneath it, but the molecular constituents of the arciform density have not been identified.

The protein bassoon has received a great deal of attention as a ribbon coorganizer (Dick et al., Reference Dick, tom Dieck, Altrock, Ammermüller, Weiler, Garner, Gundelfinger and Brandstätter2003; tom Dieck et al., Reference tom Dieck, Altrock, Kessels, Qualmann, Regus, Brauner, Fejtová, Bracko, Gundelfinger and Brandstätter2005; Frank et al., Reference Frank, Rutherford, Strenzke, Neef, PangršiČ, Khimich, Fetjova, Gundelfinger, Liberman, Harke, Bryan, Lee, Egner, Riedel and Moser2010; Regus-Leidig et al., Reference Regus-Leidig, tom Dieck and Brandstätter2010), although it is not localized exclusively to ribbon synapses (tom Dieck et al., Reference tom Dieck, Sanmartí-Vila, Langnaese, Richter, Kindler, Soyke, Wex, Smalla, Kämpf, Fränzer, Stumm, Garner and Gundelfinger1998; Richter et al., Reference Richter, Langnaese, Kreutz, Olias, Zhai, Scheich, Garner and Gundelfinger1999; Altrock et al., Reference Altrock, tom Dieck, Sokolov, Meyer, Sigler, Brakebusch, Fässler, Richter, Boeckers, Potschka, Brandt, Löscher, Grimberg, Dresbach, Hempelmann, Hassan, Balschun, Frey, Brandstätter, Garner, Rosenmund and Gundelfinger2003; Hallermann et al., Reference Hallermann, Fejtova, Schmidt, Weyhersmüller, Silver, Gundelfinger and Eilers2010). Bassoon is a large 420 KD protein that comigrates with ribeye in precursor spheres during development (Regus-Leidig et al., Reference Regus-Leidig, tom Dieck, Specht, Meyer and Brandstätter2009) and has been localized to the base of the synaptic ribbon (Brandstätter et al., Reference Brandstätter, Fletcher, Garner, Gundelfinger and Wässle1999; tom Dieck et al., Reference tom Dieck, Altrock, Kessels, Qualmann, Regus, Brauner, Fejtová, Bracko, Gundelfinger and Brandstätter2005). Bassoon may function as a ribbon-anchoring protein, but it is not clear if it is directly associated with the arciform density (Zanazzi & Matthews, Reference Zanazzi and Matthews2009). Bassoon knockout animals have detached ribbons, deformed Ca²⁺ channel clusters, and impaired vesicle attachment to the membrane in both the retina (Dick et al., Reference Dick, tom Dieck, Altrock, Ammermüller, Weiler, Garner, Gundelfinger and Brandstätter2003; tom Dieck et al., Reference tom Dieck, Altrock, Kessels, Qualmann, Regus, Brauner, Fejtová, Bracko, Gundelfinger and Brandstätter2005) and auditory hair cells (Khimich et al., Reference Khimich, Nouvian, Pujol, Tom Dieck, Egner, Gundelfinger and Moser2005; Frank et al., Reference Frank, Rutherford, Strenzke, Neef, PangršiČ, Khimich, Fetjova, Gundelfinger, Liberman, Harke, Bryan, Lee, Egner, Riedel and Moser2010). In the retina, bassoon knockout mice exhibit diminished ERG b-waves. In hair cells of bassoon knockout mice, fast synchronous release is inhibited more strongly than slower sustained release (Khimich et al., Reference Khimich, Nouvian, Pujol, Tom Dieck, Egner, Gundelfinger and Moser2005), suggesting a role for nonribbon sites in maintaining tonic neurotransmission in these cells. At ribbon and nonribbon synapses, bassoon mutants also show defects in vesicle replenishment, particularly for fast refilling (Frank et al., Reference Frank, Rutherford, Strenzke, Neef, PangršiČ, Khimich, Fetjova, Gundelfinger, Liberman, Harke, Bryan, Lee, Egner, Riedel and Moser2010; Hallermann et al., Reference Hallermann, Fejtova, Schmidt, Weyhersmüller, Silver, Gundelfinger and Eilers2010). Bassoon and ribeye often colocalize with the protein piccolo (Cases-Langhoff et al., Reference Cases-Langhoff, Voss, Garner, Appeltauer, Takei, Kindler, Veh, De Camilli, Gundelfinger and Garner1996), in both initial precursor spheres (Dresbach et al., Reference Dresbach, Torres, Wittenmayer, Altrock, Zamorano, Zuschratter, Nawrotzki, Ziv, Garner and Gundelfinger2006; Regus-Leidig et al., Reference Regus-Leidig, tom Dieck and Brandstätter2010) and fully functional ribbon-associated active zones (Dick et al., Reference Dick, Hack, Altrock, Garner, Gundelfinger and Brandstätter2001; tom Dieck et al., Reference tom Dieck, Altrock, Kessels, Qualmann, Regus, Brauner, Fejtová, Bracko, Gundelfinger and Brandstätter2005). Bassoon and piccolo share a similar structure and may share similar functions (Takao-Rikitsu et al., Reference Takao-Rikitsu, Mochida, Inoue, Deguchi-Tawarada, Inoue, Ohtsuka and Takai2004; Mukherjee et al., Reference Mukherjee, Yang, Gerber, Kwon, Ho, Castillo, Liu and Südhof2010).

Cytomatrix at the active zone-associated structural protein (CAST) helps to organize the active zone at many synapses by interacting with bassoon, piccolo, RIM1, and RIM2, as well as indirectly with Munc13 (Hida & Ohtsuka, Reference Hida and Ohtsuka2010). CAST is concentrated at the base of the ribbon along with RIM2, piccolo, and bassoon (Takao-Rikitsu et al., Reference Takao-Rikitsu, Mochida, Inoue, Deguchi-Tawarada, Inoue, Ohtsuka and Takai2004; Deguchi-Tawarada et al., Reference Deguchi-Tawarada, Inoue, Takao-Rikitsu, Inoue, Kitajima, Ohtsuka and Takai2006). RIM proteins interact with many of the key proteins in release including synaptotagmin 1, SNAP-25, Rab3A, Munc13, and Ca_V channels (Matteoli et al., Reference Matteoli, Takei, Cameron, Hurlbut, Johnston, Südhof, Jahn and De Camilli1991; Geppert et al., Reference Geppert, Goda, Stevens and Südhof1997; Wang et al., Reference Wang, Okamoto, Schmitz, Hofmann and Südhof1997; von Kriegstein et al., Reference Von Kriegstein, Schmitz, Link and Südhof1999; Coppola et al., Reference Coppola, Magnin-Luthi, Perret-Menoud, Gattesco, Schiavo and Regazzi2001; Schoch et al., Reference Schoch, Castillo, Jo, Mukherjee, Geppert, Wang, Schmitz, Malenka and Südhof2002; Deguchi-Tawarada et al., Reference Deguchi-Tawarada, Inoue, Takao-Rikitsu, Inoue, Kitajima, Ohtsuka and Takai2006; Gracheva et al., Reference Gracheva, Hadwiger, Nonet and Richmond2008; Kaeser et al., Reference Kaeser, Deng, Wang, Dulubova, Liu, Rizo and Südhof2011).

RIM1 and RIM2 are differentially located with RIM1 found along the face of the ribbon and RIM2 clustered at the base of the ribbon (tom Dieck et al., Reference tom Dieck, Altrock, Kessels, Qualmann, Regus, Brauner, Fejtová, Bracko, Gundelfinger and Brandstätter2005). ELKS has a high degree of homology to CAST and shares similar binding partners including bassoon and RIM1 (Inoue et al., Reference Inoue, Deguchi-Tawarada, Takao-Rikitsu, Inoue, Kitajima, Ohtsuka and Takai2006), but it is not concentrated as tightly at the ribbon base (Deguchi-Tawarada et al., Reference Deguchi-Tawarada, Inoue, Takao-Rikitsu, Inoue, Kitajima, Ohtsuka and Takai2006). This suggests that RIM2 may interact with CAST near the base of the ribbon, whereas interactions between RIM1 and ELKS may predominate further away from the base. CAST, ELKS, bassoon, piccolo, and RIM all appear to interact with an N-terminal region of Munc13. Interactions among these proteins may form an important node for organizing the synaptic active zone (Wang et al., Reference Wang, Hu, Zieba, Neumann, Kasper-Sonnenberg, Honsbein, Hultqvist, Conze, Witt, Limbach, Geitmann, Danielson, Kolarow, Niemann, Lessmann and Kilimann2009; Hida & Ohtsuka, Reference Hida and Ohtsuka2010).

Interactions between RIM proteins and the C-terminal region of Ca_V channel β subunits promote the clustering Ca_V channels close to synaptic vesicles (Coppola et al., Reference Coppola, Magnin-Luthi, Perret-Menoud, Gattesco, Schiavo and Regazzi2001; Kiyonaka et al., Reference Kiyonaka, Wakamori, Miki, Uriu, Nonaka, Bito, Beedle, Mori, Hara, De Waard, Kanagawa, Itakura, Takahashi, Campbell and Mori2007; Gebhart et al., Reference Gebhart, Juhasz-Vedres, Zuccotti, Brandt, Engel, Trockenbacher, Kaur, Obermair, Knipper, Koschak and Striessnig2010; Han et al., Reference Han, Kaeser, Südhof and Schneggenburger2011; Kaeser et al., Reference Kaeser, Deng, Wang, Dulubova, Liu, Rizo and Südhof2011). Interactions between Ca_V channels and RIM proteins can also be promoted by intermediary RIM-binding proteins (Hibino et al., Reference Hibino, Pironkova, Onwumere, Vologodskaia, Hudspeth and Lesage2002).

Ca_V β subunits possess SH3-HOOK-GK domains that can potentially mediate multiple binding interactions with cytoskeletal elements, classifying them as membrane-associated guanylate kinase (MAGUK) proteins (Chen et al., Reference Chen, Li, Zhang, He, Yamada, Fitzmaurice, Shen, Zhang, Tong and Yang2004; Takahashi et al., Reference Takahashi, Miriyala and Colecraft2004; Buraei & Yang, Reference Buraei and Yang2010). Although Ca_V channel β subunits are MAGUKs with homology to other scaffolding proteins like PSD-95, their interactions with other cytoskeletal proteins are limited because the guanylate kinase domain is normally shielded by interactions with the α₁ pore-forming subunit and the SH3 domain is shielded by other domains of the β subunit (Buraei & Yang, Reference Buraei and Yang2010).

In addition to potential cytoskeletal interactions with Ca_V β subunits, the pore-forming α₁ subunit of Ca_V1.3 channels has a carboxyterminal sequence that recognizes class I PDZ domains (Zhang et al., Reference Zhang, Maximov, Fu, Xu, Tang, Tkatch, Surmeier and Bezprozvanny2005). Ca_V1.3 channels in photoreceptors can interact through this sequence with PDZ domains on the Usher proteins whirlin (Kersten et al., Reference Kersten, van Wijk, van Reeuwijk, van der Zwaag, Märker, Peters, Katsanis, Wolfrum, Keunen, Roepman and Kremer2010) and harmonin (Gregory et al., Reference Gregory, Bryan, Pangršič, Calin-Jageman, Moser and Lee2011). This recognition sequence may also facilitate interactions between Ca²⁺ channels and other PDZ domain proteins (Nourry et al., Reference Nourry, Grant and Borg2003; Feng & Zhang, Reference Feng and Zhang2009).

Unc119 is located near the base of the ribbon where it plays a role in endocytosis (Karim et al., Reference Karim, Vepachedu, Gorska and Alam2010) and interacts with both ribeye and the calcium-binding protein CaBP4 (Higashide et al., Reference Higashide, McLaren and Inana1998; Alpadi et al., Reference Alpadi, Magupalli, Käppel, Köblitz, Schwarz, Seigel, Sung and Schmitz2008; Haeseleer, Reference Haeseleer2008). CaBP4 can in turn interact with Ca_V1.4 or Ca_V1.3 channels (Haeseleer et al., Reference Haeseleer, Imanishi, Maeda, Possin, Maeda, Lee, Rieke and Palczewski2004; Maeda et al., Reference Maeda, Lem, Palczewski and Haeseleer2005; Yang et al., Reference Yang, Alseikhan, Hiel, Grant, Mori, Yang, Fuchs and Yue2006), and these interactions may further help to localize Ca_V channels near the base of the ribbon.

Endocytic proteins like dynamin, amphiphysin, and clathrin are also expressed at ribbon synapses (Ullrich & Südhof, Reference Ullrich and Südhof1994: Sherry & Heidelberger, Reference Sherry and Heidelberger2005). An amphiphysin splice variant has been identified in retinal ribbon synapses (Hosoya et al., Reference Hosoya, Tsutsui and Tsutsui2004). Synaptojanin is a polyphosphoinositide phosphatase that plays a role in clathrin-mediated endocytosis (Song & Zinsmaier, Reference Song and Zinsmaier2003; Chang-Ileto et al., Reference Chang-Ileto, Frere, Chan, Voronov, Roux and Di Paolo2011). Synaptojanin antibodies label cone terminals more prominently than rod terminals (Holzhausen et al., Reference Holzhausen, Lewis, Cheong and Brockerhoff2009), and loss of synaptojanin produces floating ribbons in cone terminals (Van Epps et al., Reference Van Epps, Hayashi, Lucast, Stearns, Hurley, De Camilli and Brockerhoff2004). Unlike cone synapses, rod synapses develop normally without synaptojanin suggesting different roles for this protein and thus perhaps different endocytic pathways in the two cell types (Holzhausen et al., Reference Holzhausen, Lewis, Cheong and Brockerhoff2009).

Exocytotic proteins

There is evidence that release from ribbon synapses of inner hair cells does not involve neuronal SNARE proteins (Nouvian et al., Reference Nouvian, Neef, Bulankina, Reisinger, Pangršič, Frank, Sikorra, Brose, Binz and Moser2011). However, photoreceptor ribbon synapses, like those of most other neurons, appear to employ SNARE proteins during vesicle fusion: synaptobrevin (VAMP1 and 2), SNAP-25, and syntaxin (Ullrich & Südhof, Reference Ullrich and Südhof1994; Brandstätter et al., Reference Brandstätter, Löhrke, Morgans and Wässle1996a,Reference Brandstätter, Wässle, Betz and Morgansb; Greenlee et al., Reference Greenlee, Swanson, Simon, Elmquist, Jacobson and Sakaguchi1996, Reference Greenlee, Roosevelt and Sakaguchi2001; von Kriegstein et al., Reference Von Kriegstein, Schmitz, Link and Südhof1999; Bergmann et al., Reference Bergmann, Grabs and Rager2000; Morgans, Reference Morgans2000; Sherry et al., Reference Sherry, Yang and Standifer2001, Reference Sherry, Mitchell, Standifer and du Plessis2006; Heidelberger et al., Reference Heidelberger, Wang and Sherry2003). The v-SNARE, synaptobrevin, is located exclusively on synaptic vesicles. Immunoelectron micrographs suggest that the t-SNARE, SNAP-25, may be present not only on the plasma membrane but also on vesicles along the ribbon (Brändstatter et al., Reference Brandstätter, Löhrke, Morgans and Wässle1996b; Morgans et al., Reference Morgans, Brandstätter, Kellerman, Betz and Wässle1996; Morgans & Brändstatter, Reference Morgans2000). It is possible that vesicular SNAP-25 may promote vesicle priming or compound fusion prior to synaptic release. Ribbon synapses also possess Munc18 proteins, which assist in assembling the SNARE complex and priming vesicles (Ullrich & Südhof, Reference Ullrich and Südhof1994). One difference between photoreceptors and most other neurons is the reliance on syntaxin 3 rather than the syntaxin 1 isoform used at many synapses (Morgans et al., Reference Morgans, Brandstätter, Kellerman, Betz and Wässle1996; Curtis et al., Reference Curtis, Doneske, Liu, Thaller, McNew and Janz2008). Additionally, retinal ribbon synapses use complexins 3 and 4 rather than complexins 1 and 2 used at most other terminals (Reim et al., Reference Reim, Wegmeyer, Brandstätter, Xue, Rosenmund, Dresbach, Hofmann and Brose2005; Zanazzi & Matthews, Reference Zanazzi and Matthews2010). In complexin 3/4 knockout mice, ∼25% of the synaptic terminals exhibit free floating spherical ribbons perhaps as a result of altered synaptic activity (Reim et al., Reference Reim, Wegmeyer, Brandstätter, Xue, Rosenmund, Dresbach, Hofmann and Brose2005).

The identity of the calcium sensor molecules that regulate exocytosis from photoreceptors is unclear. Experiments on nonmammalian rods and cones show that the sensor exhibits an unusually high affinity for Ca²⁺ with a threshold of 400 nM and low cooperativity of approximately two Ca²⁺ ions (Rieke & Schwartz, Reference Rieke and Schwartz1996; Thoreson et al., Reference Thoreson, Rabl, Townes-Anderson and Heidelberger2004; Sheng et al., Reference Sheng, Choi, Dharia, Li, Sterling and Kramer2007; Duncan et al., Reference Duncan, Rabl, Gemp, Heidelberger and Thoreson2010). This is quite different from release at synapses employing synaptotagmin 1, which show a cooperativity of five Ca²⁺ ions and a requirement for much higher Ca²⁺ levels (Heidelberger et al., Reference Heidelberger, Heinemann, Neher and Matthews1994; Bollmann et al., Reference Bollmann, Sakmann and Borst2000; Schneggenburger & Neher, Reference Schneggenburger and Neher2000; Beutner et al., Reference Beutner, Voets, Neher and Moser2001). Kreft et al. (Reference Kreft, Krizaj, Grilc and Zorec2003) found that salamander rod terminals could be labeled with an antibody to synaptotagmin 1, but other synaptotagmin 1 antibodies do not label terminals of photoreceptors in salamander or goldfish (Berntson & Morgans, Reference Berntson and Morgans2003; Heidelberger et al., Reference Heidelberger, Wang and Sherry2003; Fox & Sanes, Reference Fox and Sanes2007). By contrast with these results in lower vertebrates, antibodies to synaptotagmin 1 consistently label photoreceptor ribbon synapses in the OPL of mammalian and chick retina (Greenlee et al., Reference Greenlee, Swanson, Simon, Elmquist, Jacobson and Sakaguchi1996; Bergmann et al., Reference Bergmann, Grabs and Rager2000; Berntson & Morgans, Reference Berntson and Morgans2003; Heidelberger et al., Reference Heidelberger, Wang and Sherry2003; von Kriegstein & Schmitz, Reference von Kriegstein and Schmitz2003; Lazzell et al., Reference Lazzell, Belizaire, Thakur, Sherry and Janz2004; Fox & Sanes, Reference Fox and Sanes2007; Wahlin et al., Reference Wahlin, Moreira, Huang, Yu and Adler2008). Antibodies for synaptotagmin 2 do not label photoreceptor terminals in chick, mouse, salamander, or goldfish, although there is evidence for labeling of terminals in ray retina (Fox & Sanes, Reference Fox and Sanes2007). In hair cells, it has been proposed that otoferlin or synaptotagmin 4 might be the calcium sensor (Roux et al., Reference Roux, Safieddine, Nouvian, Grati, Simmler, Bahloul, Perfettini, Le Gall, Rostaing, Hamard, Triller, Avan, Moser and Petit2006; Johnson et al., Reference Johnson, Franz, Kuhn, Furness, Rüttiger, Münkner, Rivolta, Seward, Herschman, Engel, Knipper and Marcotti2010). Otoferlin is absent from photoreceptor ribbon synapses (Uthaiah & Hudspeth, Reference Uthaiah and Hudspeth2010); the distribution of synaptotagmin 4 in the retina has not been examined.

Cytoskeletal components

Ribeye and ribbon-associated proteins interact extensively with the intracellular cytoskeleton. Much of the early work elucidating cytoskeletal networks in photoreceptors was performed using species whose rods and cones show photosensitive contractile motion (Burnside, Reference Burnside1986; Nagle et al., Reference Nagle, Okamoto, Taggart and Burnside1986) or regenerative properties that require cytoskeletal-dependent trafficking (Mandell et al., Reference Mandell, MacLeish and Townes-Anderson1993; Vecino & Avila, Reference Vecino and Avila2001; Hasegawa et al., Reference Hasegawa, Hisatomi, Yamamoto, Ono and Tokunaga2007). These studies focused more on changes in cellular morphology than on specific molecular interactions, but they offered insight into the subcellular organization of the cytoskeleton and generally agreed that there are moderate levels of actin within the synaptic terminals of both rod and cone photoreceptors (Nagle et al., Reference Nagle, Okamoto, Taggart and Burnside1986; Schmitz & Drenckhahn, Reference Schmitz and Drenckhahn1993; Schmitz et al., Reference Schmitz, Holbach and Drenckhahn1993; Katsumata et al., Reference Katsumata, Ohara, Tamaki, Niimura, Naganuma, Watanabe and Sakagami2009). Actin and microtubule networks in photoreceptors help to stabilize active zone components and to aid in cargo trafficking to and from the synapse (Schmitz & Drenckhahn, Reference Schmitz and Drenckhahn1997; Zanazzi & Matthews, Reference Zanazzi and Matthews2009).

Interactions between actin and various binding partners help to sculpt pre- and postsynaptic structure (Doussau & Augustine, Reference Doussau and Augustine2000; Hotulainen & Hoogenraad, Reference Hotulainen and Hoogenraad2010). For example, actin interacts with dystrophin and closely related spectrin proteins, all of which are found in photoreceptor terminals (Lazarides et al., Reference Lazarides, Nelson and Kasamatsu1984; Isayama et al., Reference Isayama, Goodman and Zagon1991; Spencer et al., Reference Spencer, Moon and Milam1991; Schmitz et al., Reference Schmitz, Holbach and Drenckhahn1993; Ueda et al., Reference Ueda, Kobayashi, Mitsui, Tsurugi, Tsukahara and Ohno1995, Reference Ueda, Gohdo and Ohno1998; Drenckhahn et al., Reference Drenckhahn, Holbach, Ness, Schmitz and Anderson1996; Blank et al., Reference Blank, Koulen and Kröger1997, Reference Blank, Koulen, Blake and Kröger1999; Morgans, Reference Morgans2000; Jastrow et al., Reference Jastrow, Koulen, Altrock and Kröger2006; Uthaiah and Hudspeth, Reference Uthaiah and Hudspeth2010). One of the more common binding partners for actin in most presynaptic terminals is synapsin, but synapsin is absent from photoreceptor terminals (Mandell et al., Reference Mandell, Townes-Anderson, Czernik, Cameron, Greengard and De Camilli1990; Koontz & Hendrickson, Reference Koontz and Hendrickson1993). Its absence at ribbon synapses is thought to explain the high mobility of synaptic vesicles at ribbon synapses (Holt et al., Reference Holt, Cooke, Neef and Lagnado2004; Rea et al., Reference Rea, Li, Dharia, Levitan, Sterling and Kramer2004). The network of actin fibers can exhibit tremendous plasticity as shown in hair cells where the entire actin cytoskeleton turns over every 48 h (Schneider et al., Reference Schneider, Belyantseva, Azevedo and Kachar2002). The regulation of actin varies among different retinal cell types but often involves Ca²⁺-dependent and Ca_V-associated processes (Fukuda et al., Reference Fukuda, Kameyama and Yamaguchi1981; Johnson & Byerly, Reference Johnson and Byerly1993; Job & Lagnado, Reference Job and Lagnado1998; Schubert & Akopian, Reference Schubert and Akopian2004, Reference Cristofanilli and Akopian2006; Oertner & Matus, Reference Oertner and Matus2005; Cristofanilli et al., Reference Cristofanilli, Mizuno and Akopian2007; Mizuno et al., Reference Mizuno, Barabas, Krizaj and Akopian2010). At photoreceptor synapses, single particle tracking studies show that individual Ca_V channels diffuse more freely after the disruption of actin with cytochalasin, raising the possibility that an actin network helps to maintain the spatial position of photoreceptor synaptic proteins (Mercer et al., Reference Mercer, Chen and Thoreson2011a).

In addition to actin, the other two primary cytoskeletal proteins, intermediate filaments and microtubules, are present in photoreceptors (Gray, Reference Gray1976; Nagle et al., Reference Nagle, Okamoto, Taggart and Burnside1986). Intermediate filaments are present in the axon and perinuclear region, whereas microtubules are found throughout the cell (Nagle et al., Reference Nagle, Okamoto, Taggart and Burnside1986). At many synapses, microtubules play an important role in vesicle delivery (Elluru et al., Reference Elluru, Bloom and Brady1995; Lin-Jones et al., Reference Lin-Jones, Parker, Wu, Knox and Burnside2003; Gonzalez-Bellido et al., Reference Gonzalez-Bellido, Wardill, Kostyleva, Meinertzhagen and Juusola2009) and, consistent with such a possibility, they can sometimes be observed close to the ribbon and ribbon-associated vesicles (Gray, Reference Gray1976; Nagle et al., Reference Nagle, Okamoto, Taggart and Burnside1986; Zanazzi & Matthews, Reference Zanazzi and Matthews2009).

Deficits in cytoskeletal and cytomatrix proteins can cause neurological defects including Usher syndrome (Williams, Reference Williams2008), which is the most common form of deaf-blindness. Clinically, there are three subtypes of Usher syndrome, caused by at least 12 identified gene loci (Kremer et al., Reference Kremer, van Wijk, Märker, Wolfrum and Roepman2006; Maerker et al., Reference Maerker, van Wijk, Overlack, Kersten, McGee, Goldmann, Sehn, Roepman, Walsh, Kremer and Wolfrum2008; Friedman et al., Reference Friedman, Schultz, Ahmed, Tsilou and Brewer2011; Richardson et al., Reference Richardson, de Monvel and Petit2011). Seven USH1 loci have been identified to date and are responsible for the most severe form of deaf-blindness syndrome (Ahmed et al., Reference Ahmed, Riazuddin, Riazuddin and Wilcox2003; Yan & Liu, Reference Yan and Liu2010). These gene regions encode for various cytomatrix molecules including the scaffold proteins harmonin (USH1C) and “scaffold protein containing ankyrin repeats/SAM domain” (USH1G), the motor protein myosin VIIa (USH1B), and the cell–cell adhesion molecules cadherin 23 (USH1D) and protocadherin 15 (USH1F) (Reiners et al., Reference Reiners, Reidel, El-Amraoui, Boëda, Huber, Petit and Wolfrum2003, Reference Reiners, Märker, Jürgens, Reidel and Wolfrum2005a, Reference Reiners, Nagel-Wolfrum, Jürgens, Märker and Wolfrum2006; Adato et al., Reference Adato, Michel, Kikkawa, Reiners, Alagramam, Weil, Yonekawa, Wolfrum, El-Amraoui and Petit2005; El-Amraoui & Petit, Reference El-Amraoui and Petit2005). Three USH2 loci encode for the scaffold proteins usherin (USH2A) and whirlin (USH2D) as well as very large G-protein-coupled receptor 1b (USH2C) (Reiners et al., Reference Reiners, van Wijk, Märker, Zimmermann, Jürgens, te Brinke, Overlack, Roepman, Knipper, Kremer and Wolfrum2005b; Yang et al., Reference Yang, Liu, Zhao, Adamian, Pawlyk, Sun, McMillan, Liberman and Li2010). The downstream product of the lone USH3 gene is the synaptic protein clarin-1 (Adato et al., Reference Adato, Vreugde, Joensuu, Avidan, Hamalainen, Belenkiy, Olender, Bonne-Tamir, Ben-Asher, Espinos, Millán, Lehesjoki, Flannery, Avraham, Pietrokovski, Sankila, Beckmann and Lancet2002; Isosomppi et al., Reference Isosomppi, Västinsalo, Geller, Heon, Flannery and Sankila2009). Many of the Usher proteins interact with one another, as well as with the actin cytoskeleton (Boëda et al., Reference Boëda, El-Amraoui, Bahloul, Goodyear, Daviet, Blanchard, Perfettini, Fath, Shorte, Reiners, Houdusse, Legrain, Wolfrum, Richardson and Petit2002; Inoue et al., Reference Inoue, Deguchi-Tawarada, Takao-Rikitsu, Inoue, Kitajima, Ohtsuka and Takai2006; Rzadzinska et al., Reference Rzadzinska, Schneider, Davies, Riordan and Kachar2004) and cell–cell adhesion molecules (Küssel-Andermann et al., Reference Küssel-Andermann, El-Amraoui, Safieddine, Nouaille, Perfettini, Lecuit, Cossart, Wolfrum and Petit2000; Reiners et al., Reference Reiners, Nagel-Wolfrum, Jürgens, Märker and Wolfrum2006). In hair cell stereocilia and the connecting cilium in photoreceptors, these protein networks appear to be important for intracellular protein trafficking and structural organization. In addition to being found in cilia, whirlin, myosin VIIa, harmonin, cadherin 23, protocadherin 15, and clarin-1 are also present in photoreceptor terminals where they likely help organize the structure of the ribbon synapse (El-Amraoui et al., Reference El-Amraoui, Sahly, Picaud, Sahel, Abitbol and Petit1996; Reiners et al., Reference Reiners, Reidel, El-Amraoui, Boëda, Huber, Petit and Wolfrum2003, Reference Reiners, Märker, Jürgens, Reidel and Wolfrum2005a; Williams et al., Reference Williams, Aleman, Lillo, Lopes, Hughes, Stone and Jacobson2009; Zallocchi et al., Reference Zallocchi, Meehan, Delimont, Askew, Garige, Gratton, Rothermund-Franklin and Cosgrove2009).

Lipid rafts

Along with cytoskeletal interactions, the distribution of lipids within the plasma membrane is a major mechanism for segregating and compartmentalizing membrane proteins (Lenne et al., Reference Lenne, Wawrezinieck, Conchonaud, Wurtz, Boned, Guo, Rigneault, He and Marguet2006). Using electron microscopy and a cholesterol-binding antibiotic, filipin, Cooper and McLaughlin (Reference Cooper and McLaughlin1984) found that photoreceptors display a high concentration of cholesterol in lipid rafts at margins of the ribbon-style active zone. Consistent with the presence of lipid rafts in photoreceptor terminals, Mercer et al. (Reference Mercer, Chen and Thoreson2011a) found that labeling of lipid raft ganglioside GM1 glycoproteins with fluorescein isothiocyanate-conjugated toxin B stained the OPL. Cholesterol-containing lipid rafts were absent from nonribbon locations in presynaptic terminals and were not observed in the postsynaptic dendrites of horizontal and bipolar cells (Cooper & McLaughlin, Reference Cooper and McLaughlin1984). P-/Q-type Ca_V channels and α₂δ Ca_V channel accessory subunits have been shown to localize preferentially to lipid rafts (Taverna et al., Reference Taverna, Saba, Rowe, Francolini, Clementi and Rosa2004; Davies et al., Reference Davies, Douglas, Hendrich, Wratten, Tran Van Minh, Foucault, Koch, Pratt, Saibil and Dolphin2006), suggesting that membrane cholesterol may assist in clustering ion channels close to vesicle release machinery (Lang et al., Reference Lang, Bruns, Wenzel, Riedel, Holroyd, Thiele and Jahn2001; Gil et al., Reference Gil, Soler-Jover, Blasi and Aguilera2004; Salaün et al., Reference Salaün, Gould and Chamberlain2005; Taverna et al., Reference Taverna, Saba, Linetti, Longhi, Jeromin, Righi, Clementi and Rosa2007; Linetti et al., Reference Linetti, Fratangeli, Taverna, Valnegri, Francolini, Cappello, Matteoli, Passafaro and Rosa2010). Consistent with such a possibility at photoreceptor synapses, depleting membrane cholesterol from photoreceptors expanded the synaptic confinement area for Ca_V channels (Mercer et al., Reference Mercer, Chen and Thoreson2011a). These results also support the hypothesis that lipid rafts act as diffusion barriers to limit the lateral mobility of membrane proteins (Renner et al., Reference Renner, Choquet and Triller2009).

Summary of protein interactions within the photoreceptor ribbon synapse

Fig. 2 illustrates the coassembly of proteins at the photoreceptor ribbon synapse. Interactions among ribeye A domains form the backbone of the ribbon (Schmitz et al., Reference Schmitz, Königstorfer and Südhof2000; Magupalli et al., Reference Magupalli, Schwarz, Alpadi, Natarajan, Seigel and Schmitz2008), whereas ribeye B domains on the outer face of the ribbon appear to link it to scaffold proteins at the base such as bassoon, piccolo, and Unc119 (Alpadi et al., Reference Alpadi, Magupalli, Käppel, Köblitz, Schwarz, Seigel, Sung and Schmitz2008). Bassoon and piccolo also interact with CAST and the homologous protein ELKS (Takao-Rikitsu et al., Reference Takao-Rikitsu, Mochida, Inoue, Deguchi-Tawarada, Inoue, Ohtsuka and Takai2004; Tokoro et al., Reference Tokoro, Higa, Deguchi-Tawarada, Inoue, Kitajima and Ohtsuka2007). The protein “hub” formed by CAST, bassoon, piccolo, RIM, and Munc13 proteins helps to cluster a number of proteins near the ribbon (Ohara-Imaizumi et al., Reference Ohara-Imaizumi, Ohtsuka, Matsushima, Akimoto, Nishiwaki, Nakamichi, Kikuta, Nagai, Kawakami, Watanabe and Nagamatsu2005; Inoue et al., Reference Inoue, Deguchi-Tawarada, Takao-Rikitsu, Inoue, Kitajima, Ohtsuka and Takai2006; Wang et al., Reference Wang, Hu, Zieba, Neumann, Kasper-Sonnenberg, Honsbein, Hultqvist, Conze, Witt, Limbach, Geitmann, Danielson, Kolarow, Niemann, Lessmann and Kilimann2009; Hida & Ohtsuka, Reference Hida and Ohtsuka2010). Contacts between SNARE proteins and Munc13 may help to localize SNARE complexes beneath the ribbon (Betz et al., Reference Betz, Okamoto, Benseler and Brose1997; Guan et al., Reference Guan, Dai and Rizo2008; Deng et al., Reference Deng, Kaeser, Xu and Südhof2011), while interactions between RIM and Munc13 with Rab3a may link this ribbon complex directly to vesicles (Dulubova et al., Reference Dulubova, Lou, Lu, Huryeva, Alam, Schneggenburger, Südhof and Rizo2005). Ca_V channels can associate with the ribbon by virtue of interactions with RIM proteins, CaBP4, and Unc119 (Hibino et al., Reference Hibino, Pironkova, Onwumere, Vologodskaia, Hudspeth and Lesage2002; Kiyonaka et al., Reference Kiyonaka, Wakamori, Miki, Uriu, Nonaka, Bito, Beedle, Mori, Hara, De Waard, Kanagawa, Itakura, Takahashi, Campbell and Mori2007; Han et al., Reference Han, Kaeser, Südhof and Schneggenburger2011; Kaeser et al., Reference Kaeser, Deng, Wang, Dulubova, Liu, Rizo and Südhof2011). Many other proteins are found at photoreceptor ribbon synapses (Uthaiah and Hudspeth, Reference Uthaiah and Hudspeth2010), and thus, interactions with other synaptic, scaffolding, and motor proteins (Muresan et al., Reference Muresan, Lyass and Schnapp1999; tom Dieck et al., Reference tom Dieck, Altrock, Kessels, Qualmann, Regus, Brauner, Fejtová, Bracko, Gundelfinger and Brandstätter2005; Katsumata et al., Reference Katsumata, Ohara, Tamaki, Niimura, Naganuma, Watanabe and Sakagami2009) are also likely to help organize Ca_V channels, synaptic vesicles, and fusion machinery at the ribbon synapse.

Fig. 2.

Protein–protein interactions at the photoreceptor ribbon synapse. The synaptic ribbon anchors to bassoon and piccolo at the terminals of rods and cones. The vertical ribbon is complexed at the base through CAST to a hub including RIM2, Rab3a, and Munc13. This synaptic node may help to position primed vesicles close to Ca_V channels and the fusion machinery (e.g., syntaxin, SNAP-25). Ca_V channels also appear to interact with the actin cytoskeleton and, by virtue of interactions with CaBP4, RIM, and Unc119, may couple directly to the ribbon itself. Kif3a and a complex of ELKS and RIM1 appear to be located at more distant sites up the vertical face of the ribbon. In Figs. 2 and 3, solid lines show direct interactions and dashed lines show putative interactions, for which direct interactions have not yet been definitively established.

The ECM

In addition to interactions with intracellular proteins and intramembrane proteins, active zone-associated proteins can interact with the ECM in the synaptic cleft. A number of different synaptic cleft proteins are important for positioning presynaptic release sites across from postsynaptic receptors (Specht & Triller, Reference Specht and Triller2008; Feng & Zhang, Reference Feng and Zhang2009; Lajoie et al., Reference Lajoie, Goetz, Dennis and Nabi2009). We review ECM proteins, both retina specific (e.g., pikachurin, retinoschisin, and nyctalopin) and otherwise, that have been shown to help organize photoreceptor synapses and their postsynaptic contacts in the OPL.

Alterations in the proteins dystrophin and dystroglycan cause muscular dystrophy and can produce visual deficits (Straub & Campbell, Reference Straub and Campbell1997; Waite et al., Reference Waite, Tinsley, Locke and Blake2009). Both dystrophin and dystroglycan are expressed at the synapses of rod and cone photoreceptors (Schmitz et al., Reference Schmitz, Holbach and Drenckhahn1993; Ueda et al., Reference Ueda, Kobayashi, Mitsui, Tsurugi, Tsukahara and Ohno1995; Drenckhahn et al., Reference Drenckhahn, Holbach, Ness, Schmitz and Anderson1996; Blank et al., Reference Blank, Koulen and Kröger1997, Reference Blank, Koulen, Blake and Kröger1999; Ueda et al., Reference Ueda, Gohdo and Ohno1998; Morgans, Reference Morgans2000; Jastrow et al., Reference Jastrow, Koulen, Altrock and Kröger2006). Futhermore, mutations in dystrophin and dystroglycan can cause reductions in the ERG b-wave (Cibis et al., Reference Cibis, Fitzgerald, Harris, Rothberg and Rupani1993; Fitzgerald et al., Reference Fitzgerald, Cibis, Giambrone and Harris1994; Pillers et al., Reference Pillers, Fitzgerald, Duncan, Rash, White, Dwinnell, Powell, Schnur, Ray, Cibis and Weleber1999). A retina-specific form of dystrophin, Dp260, is localized to photoreceptor synapses (D’Souza et al., Reference D’Souza, Nguyen, Morris, Karges, Pillers and Ray1995), and reductions in this protein cause a selective loss of dystroglycan from the OPL (Kameya et al., Reference Kameya, Araki, Katsuki, Mizota, Adachi, Nakahara, Nonaka, Sakuragi, Takeda and Nabeshima1997). Dystroglycan precursor proteins are cleaved into two subunits: β-dystroglycan and α-dystroglycan. α-dystroglycan is a heavily glycosylated extracellular protein that interacts with multiple ECM partners including β-dystroglycan, pikachurin, fibulin, and laminin (Ibraghimov-Beskrovnaya et al., Reference Ibraghimov-Beskrovnaya, Ervasti, Leveille, Slaughter, Sernett and Campbell1992; Talts et al., Reference Talts, Andac, Göhring, Brancaccio and Timpl1999; Sato et al., Reference Sato, Omori, Katoh, Kondo, Kanagawa, Miyata, Funabiki, Koyasu, Kajimura, Miyoshi, Sawai, Kobayashi, Tani, Toda, Usukura, Tano, Fujikado and Furukawa2008). In addition to binding to α-dystroglycan outside the cell, β-dystroglycan spans the plasma membrane and binds to dystrophin inside the cell. Dystrophin in turn interacts with the actin cytoskeleton. Disruption of β-dystroglycan causes changes in K⁺ channel clustering in Müller cells but does not appear to alter dystrophin localization in the outer retina or cause changes in retinal lamination patterns (Satz et al., Reference Satz, Philp, Nguyen, Kusano, Lee, Turk, Riker, Hernández, Weiss, Anderson, Mullins, Moore, Stone and Campbell2009). By contrast, as discussed below, impaired interactions between α-dystroglycan and pikachurin produce significant outer retinal defects.

The ECM protein pikachurin interacts with glycosylated α-dystroglycan at photoreceptor terminals (Sato et al., Reference Sato, Omori, Katoh, Kondo, Kanagawa, Miyata, Funabiki, Koyasu, Kajimura, Miyoshi, Sawai, Kobayashi, Tani, Toda, Usukura, Tano, Fujikado and Furukawa2008; Kanagawa et al., Reference Kanagawa, Omori, Sato, Kobayashi, Miyagoe-Suzuki, Takeda, Endo, Furukawa and Toda2010; Hu et al., Reference Hu, Li, Zhang and Yu2011). The presence of pikachurin is critical for establishing contacts between rods and bipolar cells (Sato et al., Reference Sato, Omori, Katoh, Kondo, Kanagawa, Miyata, Funabiki, Koyasu, Kajimura, Miyoshi, Sawai, Kobayashi, Tani, Toda, Usukura, Tano, Fujikado and Furukawa2008). Pikachurin knockout mice yield viable offspring but show morphological defects in OPL organization (Kanagawa et al., Reference Kanagawa, Omori, Sato, Kobayashi, Miyagoe-Suzuki, Takeda, Endo, Furukawa and Toda2010) and blunted ERG responses (Sato et al., Reference Sato, Omori, Katoh, Kondo, Kanagawa, Miyata, Funabiki, Koyasu, Kajimura, Miyoshi, Sawai, Kobayashi, Tani, Toda, Usukura, Tano, Fujikado and Furukawa2008). Hypoglycosylation of α-dystroglycan can impede dystroglycan–pikachurin interactions, causing defects in the OPL similar to those observed in pikachurin knockout mice (Kanagawa et al., Reference Kanagawa, Omori, Sato, Kobayashi, Miyagoe-Suzuki, Takeda, Endo, Furukawa and Toda2010; Hu et al., Reference Hu, Li, Zhang and Yu2011). Although a postsynaptic protein partner for pikachurin has not been identified, these data suggest that pikachurin provides an intermediary tether between the terminal of a photoreceptor and dendrite of a bipolar cell.

Laminin isoforms have been implicated in the functional organization of the OPL and photoreceptor outer segment layer (Libby et al., Reference Libby, Lavallee, Balkema, Brunken and Hunter1999; Biehlmaier et al., Reference Biehlmaier, Makhankov and Neuhauss2007). Laminin interacts with multiple ECM proteins including α-dystroglycan and retinoschisin (Claudepierre et al., Reference Claudepierre, Manglapus, Marengi, Radner, Champliaud, Tasanen, Bruckner-Tuderman, Hunter and Brunken2005; Steiner-Champliaud et al., Reference Steiner-Champliaud, Sahel and Hicks2006). Seven laminin subtypes have been described in the retina (Libby et al., Reference Libby, Champliaud, Claudepierre, Xu, Gibbons, Koch, Burgeson, Hunter and Brunken2000), and mutations of at least two laminin subtypes result in floating ribbons (Biehlmaier et al., Reference Biehlmaier, Makhankov and Neuhauss2007).

Retinoschisin is a secreted retina-specific OPL matrix protein, and mutations cause the retinopathy X-linked retinoschisis (Grayson et al., Reference Grayson, Reid, Ellis, Rutherford, Sowden, Yates, Farber and Trump2000; Tantri et al., Reference Tantri, Vrabec, Cu-Unjieng, Frost, Annesley and Donoso2004; Vijayasarathy et al., Reference Vijayasarathy, Takada, Zeng, Bush and Sieving2008). This protein is a large disulfide-linked ECM organizing protein, with a critical adhesion site called the discoidin domain. Defects in the discoidin domain of retinoschisin disrupt trafficking and secretion (Wu & Molday, Reference Wu and Molday2003) and are thought to contribute to developmentally dependent changes in retina morphology and subsequent retinal dysfunction (Weber et al., Reference Weber, Schrewe, Molday, Gehrig, White, Seeliger, Jaissle, Friedburg, Tamm and Molday2002; Takada et al., Reference Takada, Fariss, Tanikawa, Zeng, Carper, Bush and Sieving2004, Reference Takada, Vijayasarathy, Zeng, Kjellstrom, Bush and Sieving2008). The discoidin domain of retinoschisin can interact with a number of binding partners: anionic phospholipids (Vijayasarathy et al., Reference Vijayasarathy, Takada, Zeng, Bush and Sieving2007; Kotova et al., Reference Kotova, Vijayasarathy, Dimitriadis, Ikonomou, Jaffe and Sieving2010), galactose (Dyka et al., Reference Dyka, Wu, Pfeifer, Molday, Grigliatti and Molday2008), Na⁺/K⁺-ATPase (Molday et al., Reference Molday, Wu and Molday2007; Shi et al., Reference Shi, Jian, Ko, Trump and Ko2009; Friedrich et al., Reference Friedrich, Stöhr, Hilfinger, Loenhardt, Schachner, Langmann and Weber2011), photoreceptor Ca_V channels (Shi et al., Reference Shi, Jian, Ko, Trump and Ko2009), laminin, alpha crystallin, and the binding partner for peanut agglutinin (Steiner-Champliaud et al., Reference Steiner-Champliaud, Sahel and Hicks2006). Like pikachurin, a postsynaptic binding partner for retinoschisin has not been identified, but retinoschisin is nevertheless essential for proper development and function of the retina (Park et al., Reference Park, Wu, Kjellstrom, Zeng, Bush, Sieving and Colosi2009; Sergeev et al., Reference Sergeev, Caruso, Meltzer, Smaoui, MacDonald and Sieving2010).

The retina-specific ECM protein nyctalopin, derived from the NYX gene, has been implicated in a form of CSNB (Bech-Hansen et al., Reference Bech-Hansen, Naylor, Maybaum, Sparkes, Koop, Birch, Bergen, Prinsen, Polomeno, Gal, Drack, Musarella, Jacobson, Young and Weleber2000; Pusch et al., Reference Pusch, Zeitz, Brandau, Pesch, Achatz, Feil, Scharfe, Maurer, Jacobi, Pinckers, Andreasson, Hardcastle, Wissinger, Berger and Meindl2000) and the mouse “no b-wave” (NYX^nob) mutant (Gregg et al., Reference Gregg, Mukhopadhyay, Candille, Ball, Pardue, McCall and Peachey2003, Reference Gregg, Kamermans, Klooster, Lukasiewicz, Peachey, Vessey and McCall2007). Analysis of ERGs in CSNB patients and NYX^nob mutant mice indicates a failure of transmission from photoreceptors to ON bipolar cells (Khan et al., Reference Khan, Kondo, Hiriyanna, Jamison, Bush and Sieving2005; Bahadori et al., Reference Bahadori, Biehlmaier, Zeitz, Labhart, Makhankov, Forster, Gesemann, Berger and Neuhauss2006; Leroy et al., Reference Leroy, Budde, Wittmer, De Baere, Berger and Zeitz2009). This is similar to effects of postsynaptic mutations in the glutamate receptor, mGluR6, or the transduction channel, TRPM1, at the ON bipolar synapse (McCall & Gregg, Reference McCall and Gregg2008; Shen et al., Reference Shen, Heimel, Kamermans, Peachey, Gregg and Nawy2009; van Genderen et al., Reference van Genderen, Bijveld, Claassen, Florijn, Pearring, Meire, McCall, Riemslag, Gregg, Bergen and Kamermans2009; Koike et al., Reference Koike, Obara, Uriu, Numata, Sanuki, Miyata, Koyasu, Ueno, Funabiki, Tani, Ueda, Kondo, Mori, Tachibana and Furukawa2010; Morgans et al., Reference Morgans, Brown and Duvoisin2010). Nyctalopin appears to target TRPM1 to the tips of ON bipolar cell dendrites, which may explain the similarity in ERG phenotypes when nyctalopin or TRPM1 are mutated (Cao et al., Reference Cao, Posokhova and Martemyanov2011; Pearring et al., Reference Pearring, Bojang, Shen, Koike, Furukawa, Nawy and Gregg2011). Like mGluR6 and TRPM1 knockout animals, nyctalopin knockout animals show normal morphology in the OPL (Ball et al., Reference Ball, Pardue, McCall, Gregg and Peachey2003). This contrasts with mutations of presynaptic proteins (e.g., Ca_V channels, bassoon, or ribeye) that lead to disorganization of the OPL, including the sprouting of ectopic synapses (McCall & Gregg, Reference McCall and Gregg2008). The relatively normal OPL structure of NYX mutants is consistent with the hypothesis that nyctalopin is more important for maintaining the dendritic organization of ON bipolar cells, whereas the disorganization of the OPL that accompanies mutations in pikachurin or retinoschisin suggests that these proteins are more important for presynaptic organization.

Crumbs (Crb), a transmembrane ECM protein, acts as a scaffold and is a critical element in the apical–basal development of photoreceptors (reviewed in Gosens et al., Reference Gosens, den Hollander, Cremers and Roepman2008). Three CRB proteins have been identified in humans and mice. Deletion or missense mutations of Crumbs genes can lead to Leber’s congenital amaurosis and retinitis pigmentosa type 12 (den Hollander et al., Reference den Hollander, ten Brink, de Kok, van Soest, van den Born, van Driel, van de Pol, Payne, Bhattacharya, Kellner, Hoyng, Westerveld, Brunner, Bleeker-Wagemakers, Deutman, Heckenlively, Cremers and Bergen1999; Lotery et al., Reference Lotery, Jacobson, Fishman, Weleber, Fulton, Namperumalsamy, Héon, Levin, Grover, Rosenow, Kopp, Sheffield and Stone2001; Meuleman et al., Reference Meuleman, van de Pavert and Wijnholds2004; van den Hurk et al., Reference van den Hurk, Rashbass, Roepman, Davis, Voesenek, Arends, Zonneveld, van Roekel, Cameron, Rohrschneider, Heckenlively, Koenekoop, Hoyng, Cremers and den Hollander2005) indicating a pivotal role in the normal assembly and function of the retina. Gain-of-function mutations also suggest that proper expression of CRB during development is required to produce the normal laminar structure of the outer retina (Fan et al., Reference Fan, Chen, Lin, Chao and Yang2003). The CRB protein assembly links photoreceptors to surrounding Müller glia at the outer limiting membrane, just beyond the outer nuclear layer (Mehalow et al., Reference Mehalow, Kameya, Smith, Hawes, Denegre, Young, Bechtold, Haider, Tepass, Heckenlively, Chang, Naggert and Nishina2003; Van de Pavert et al., Reference van de Pavert, Kantardzhieva, Malysheva, Meuleman, Versteeg, Levelt, Klooster, Geiger, Seeliger, Rashbass, Le Bivic and Wijnholds2004). CRB1, CRB2, and CRB3 bind directly to protein 4.1 (EBP41)-L5 and MPP5 family to maintain neural–glial connectivity at adherens junctions in the outer limiting membrane (Knust & Bossinger, Reference Knust and Bossinger2002; Meuleman et al., Reference Meuleman, van de Pavert and Wijnholds2004; van de Pavert et al., Reference van de Pavert, Kantardzhieva, Malysheva, Meuleman, Versteeg, Levelt, Klooster, Geiger, Seeliger, Rashbass, Le Bivic and Wijnholds2004). At photoreceptor synapses, there is evidence for CRB2, CRB3, MPP4, MPP5, and Veli3 expression but not CRB1 expression (van de Pavert et al., Reference van de Pavert, Kantardzhieva, Malysheva, Meuleman, Versteeg, Levelt, Klooster, Geiger, Seeliger, Rashbass, Le Bivic and Wijnholds2004; Kantardzhieva et al., Reference Kantardzhieva, Gosens, Alexeeva, Punte, Versteeg, Krieger, Neefjes-Mol, den Hollander, Letteboer, Klooster, Cremers, Roepman and Wijnholds2005, Reference Kantardzhieva, Alexeeva, Versteeg and Wijnholds2006; Stöhr et al., Reference Stöhr, Molday, Molday, Weber, Biedermann, Reichenbach and Krämer2005; Aartsen et al., Reference Aartsen, Kantardzhieva, Klooster, van Rossum, van de Pavert, Versteeg, Cardozo, Tonagel, Beck, Tanimoto, Seeliger and Wijnholds2006). Among other functions, these proteins are thought to regulate the synaptic localization of PMCA proteins in rods (Yang et al., Reference Yang, Pawlyk, Wen, Adamian, Soloviev, Michaud, Zhao, Sandberg, Makino and Li2007).

Despite its name, PSD-95 is not limited to postsynaptic locations but can also be found presynaptically in terminals of photoreceptors (Koulen et al., Reference Koulen, Fletcher, Craven, Bredt and Wässle1998). PSD-95 contains PDZ domains that can interact with interact with a number of proteins (Nourry et al., Reference Nourry, Grant and Borg2003; Feng and Zhang, Reference Feng and Zhang2009). In photoreceptor terminals, PSD-95 has been shown to interact with Crumbs-related proteins (Aartsen et al., Reference Aartsen, Kantardzhieva, Klooster, van Rossum, van de Pavert, Versteeg, Cardozo, Tonagel, Beck, Tanimoto, Seeliger and Wijnholds2006), PMCA (Aartsen et al., Reference Aartsen, Arsanto, Chauvin, Vos, Versteeg, Cardozo, Bivic and Wijnholds2009), and the putative calcium-activated chloride channel, TMEM16B (Stöhr et al., Reference Stöhr, Heisig, Benz, Schöberl, Milenkovic, Strauss, Aartsen, Wijnholds, Weber and Schulz2009). PMCA and calcium-activated chloride channels are not clustered tightly near the synaptic ribbon but distributed more diffusely throughout photoreceptor terminals (Morgans et al., Reference Morgans, El Far, Berntson, Wässle and Taylor1998; Mercer et al., Reference Mercer, Rabl, Riccardi, Brecha, Stella and Thoreson2011b). PSD-95 may thus be more important for organizing proteins in surrounding parts of the synaptic terminal, whereas other proteins discussed earlier (e.g.,RIM and Munc13) may be more important for organizing proteins near the ribbon.

Yamagata and Sanes (Reference Yamagata and Sanes2010) showed evidence for a transsynaptic scaffold in photoreceptors involving MAGI proteins. Like PSD-95, MAGI proteins contain multiple PDZ domains (Nourry et al., Reference Nourry, Grant and Borg2003). MAGI proteins in the presynaptic terminals of photoreceptors appear to be linked across the synaptic cleft by extracellular Sidekick-2 proteins and cadherins to MAGI proteins in postsynaptic dendrites (Yamagata et al., Reference Yamagata, Weiner and Sanes2002; Yamagata & Sanes, Reference Yamagata and Sanes2010). Sidekick-2 may be retina specific since homologous transsynaptic scaffold proteins have not yet been described at other synapses.

Summary of protein interactions with the ECM at the photoreceptor ribbon synapse

Fig. 3 summarizes various extracellular protein–protein interactions across the OPL. As described above, PSD-95 in photoreceptor terminals can link to PMCA (Aartsen et al., Reference Aartsen, Arsanto, Chauvin, Vos, Versteeg, Cardozo, Bivic and Wijnholds2009), TMEM16B calcium-activated chloride channels (Stöhr et al., Reference Stöhr, Heisig, Benz, Schöberl, Milenkovic, Strauss, Aartsen, Wijnholds, Weber and Schulz2009), and the Crumbs complex (Yang et al., Reference Yang, Pawlyk, Wen, Adamian, Soloviev, Michaud, Zhao, Sandberg, Makino and Li2007). Whereas RIM and Munc13 proteins appear to be particularly important for organizing active zone proteins close to the ribbon (Fig. 2), PSD-95 may be more important for organizing proteins in neighboring nonribbon regions of the terminal. By virtue of its interactions with laminin, Ca_V channels, and Na⁺/K⁺ ATPases, retinoschisin may be an important node for organizing ECM proteins (Molday et al., Reference Molday, Wu and Molday2007; Shi et al., Reference Shi, Jian, Ko, Trump and Ko2009; Friedrich et al., Reference Friedrich, Stöhr, Hilfinger, Loenhardt, Schachner, Langmann and Weber2011). Laminin and pikachurin can also interact with the dystroglycan/dystrophin complex (Claudepierre et al., Reference Claudepierre, Manglapus, Marengi, Radner, Champliaud, Tasanen, Bruckner-Tuderman, Hunter and Brunken2005; Kanagawa et al., Reference Kanagawa, Omori, Sato, Kobayashi, Miyagoe-Suzuki, Takeda, Endo, Furukawa and Toda2010). Other links among ECM proteins remain to be discovered, such as binding partners for nyctalopin and Crumbs complex proteins.

Fig. 3.

Structural organization and protein interactions across the OPL. There appear to be at least two major nodes of protein–protein interactions in the ECM within the OPL. The Sidekick-2 transsynaptic scaffold attaches MAGI proteins in apposed cells, with putative lateral presynaptic interactions to PSD-95. PSD-95 also appears to anchor the Cl(Ca) channel TMEM16B and PMCA extrusion pump at nonribbon sites in the terminal. The second hub of scaffolding interactions occurs between a transmembrane dystroglycan complex and the retina-specific ECM proteins pikachurin and retinoschisin. These complexes are linked through synaptic laminin and cytoplasmic actin, while also attaching to the extracellular face of transmembrane Ca_V channels and the Na⁺/K⁺-ATPase.

Dynamic changes in ribbon proteins

The need to maintain efficient and reliable neuron-to-neuron communication would appear to dictate a relatively fixed arrangement of calcium channels, vesicle release sites, and postsynaptic receptors at many synapses, including photoreceptor synapses. The many interactions among cytoskeletal, intramembrane, ribbon-associated, and ECM proteins described above also suggest a static and heavily reinforced structure. However, despite these expectations, there is evidence for a surprising degree of structural plasticity at photoreceptor ribbon synapses, some of which operates on a very rapid time scale.

As mentioned above, mutations in a number of different presynaptic proteins can cause neurodegenerative changes that are accompanied by changes in ribbon structure. For example, mutations in Ca_V1.4, bassoon, complexin, synaptojanin, and laminin can produce floating ribbons (Dick et al., Reference Dick, tom Dieck, Altrock, Ammermüller, Weiler, Garner, Gundelfinger and Brandstätter2003; Reim et al., Reference Reim, Wegmeyer, Brandstätter, Xue, Rosenmund, Dresbach, Hofmann and Brose2005; tom Dieck et al., Reference tom Dieck, Altrock, Kessels, Qualmann, Regus, Brauner, Fejtová, Bracko, Gundelfinger and Brandstätter2005; Chang et al., Reference Chang, Heckenlively, Bayley, Brecha, Davisson, Hawes, Hirano, Hurd, Ikeda, Johnson, McCall, Morgans, Nusinowitz, Peachey, Rice, Vessey and Gregg2006; Biehlmaier et al., Reference Biehlmaier, Makhankov and Neuhauss2007). Mutations in tubby-like protein 1 (TULP1), which underlies a form of autosomal recessive retinitis pigmentosa, also causes changes in the structure of photoreceptor ribbons (Grossman et al., Reference Grossman, Pauer, Narendra, Peachey and Hagstrom2009). TULP1 appears to play a role in intracellular membrane trafficking but its role in synaptic function is unclear. Additionally, mutations in cysteine string protein alpha, a chaperone protein that prevents SNAP-25 degradation and SNARE complex formation (Sharma et al., Reference Sharma, Burré and Südhof2011), cause photoreceptor degeneration accompanied by floating ribbons and the appearance of electron-dense aggregates (Schmitz et al., Reference Schmitz, Tabares, Khimich, Strenzke, de la Villa-Polo, Castellano-Muñoz, Bulankina, Moser, Fernández-Chacón and Südhof2006). In addition to changes in ribbon structure, mutations in presynaptic Ca_V channel subunits (α_1F, β₂α, α₂δ₄), and the calcium channel-associated protein CaBP4 can also promote the extension of dendritic processes from bipolar and horizontal cells into the outer nuclear layer and formation of ectopic synapses (Haeseleer et al., Reference Haeseleer, Imanishi, Maeda, Possin, Maeda, Lee, Rieke and Palczewski2004; McCall & Gregg, Reference McCall and Gregg2008).

More surprising than the ability of mutations and neurodegenerative changes to induce long-term structural changes, ribbons in rods and cones can alter their structure reversibly on a diurnal basis (Abe & Yamamoto, Reference Abe and Yamamoto1984; Vollrath & Spiwoks-Becker, Reference Vollrath and Spiwoks-Becker1996; Adly et al., Reference Adly, Spiwoks-Becker and Vollrath1999; Balkema et al., Reference Balkema, Cusick and Nguyen2001; Spiwoks-Becker et al., Reference Spiwoks-Becker, Glas, Lasarzik and Vollrath2004). At night, rod ribbons form plate-like structures, but in daytime, ribbons become much shorter and are associated with adjacent electron-dense spherical bodies. In the transition between these two forms during crepuscular conditions, the ribbons often assume a club-like appearance (Abe & Yamamoto, Reference Abe and Yamamoto1984; Vollrath & Spiwoks-Becker, Reference Vollrath and Spiwoks-Becker1996; Adly et al., Reference Adly, Spiwoks-Becker and Vollrath1999). It has recently been reported that these differences are much more pronounced in C57Bl/6J mice than Balb/cJ mice (Fuchs et al., Reference Fuchs, Sendelbeck and Brandstätter2011). Diurnal changes in ribbon shape appear to be driven by light, not circadian rhythms (Spiwoks-Becker et al., Reference Spiwoks-Becker, Glas, Lasarzik and Vollrath2004). Diurnal structural changes have also been observed in ribbons from pineal photoreceptors (Kurumado & Mori, Reference Kurumado and Mori1977; Kikuchi et al., Reference Kikuchi, Chiba and Aoki2000) and retinal bipolar cells (Hull et al., Reference Hull, Studholme, Yazulla and von Gersdorff2006). The finding that heterotypic interactions between A and B domains of ribeye are inhibited by NAD(H) has led to the proposal that metabolically driven changes in NAD(H) levels might regulate diurnal changes in ribbon structure (Magupalli et al., Reference Magupalli, Schwarz, Alpadi, Natarajan, Seigel and Schmitz2008).

Ribbons are also capable of dynamic movements on very short time scales. Bipolar cell ribbons labeled with fluorescently conjugated ribeye-binding peptide can exhibit both small (Zenisek et al., Reference Zenisek, Horst, Merrifield, Sterling and Matthews2004; Mercer et al., Reference Mercer, Chen and Thoreson2011a) and occasional large rapid excursions through the cell (Zenisek et al., Reference Zenisek, Horst, Merrifield, Sterling and Matthews2004).

Diurnal changes in postsynaptic processes have been described. However, these appear to occur only in fish retina where exposure to light stimulates the tips of horizontal cell dendrites (known as spinules) to extend into the cone pedicle and darkness causes them to retract (reviewed by Wagner & Djamgoz, Reference Wagner and Djamgoz1993).

The possibility of rapid changes in the arrangement of presynaptic proteins has been investigated by visualizing the movements of individual Ca_V channels labeled with brightly fluorescent quantum dots (Mercer et al., Reference Mercer, Chen and Thoreson2011a). Streptavidin-coated quantum dots, bound through a biotinylated secondary antibody, were attached to the extracellular domain of the α₂δ₄ accessory subunit of L-type Ca_V channels in photoreceptors. Even though Ca_V channels interact with multiple proteins, single particle tracking experiments show that individual Ca_V channels are continuously in motion within the plasma membrane of photoreceptors (Mercer et al., Reference Mercer, Chen and Thoreson2011a). Movements of Ca_V channels are confined to a small region near the ribbon that roughly matches the size of the active zone, consistent with evidence that Ca_V channels cluster beneath the ribbon in photoreceptors and hair cells (Raviola & Gilula, Reference Raviola and Gilula1975; tom Dieck et al., Reference tom Dieck, Altrock, Kessels, Qualmann, Regus, Brauner, Fejtová, Bracko, Gundelfinger and Brandstätter2005; Frank et al., Reference Frank, Rutherford, Strenzke, Neef, PangršiČ, Khimich, Fetjova, Gundelfinger, Liberman, Harke, Bryan, Lee, Egner, Riedel and Moser2010). Some of the Ca_V channel movement may reflect movement of the overlying ribbon but ribbon movements are much smaller than Ca_V channel movements (Mercer et al., Reference Mercer, Chen and Thoreson2011a).

Depolarization-evoked fusion of vesicles at the base of the ribbon can abruptly increase Ca_V channel movements, propelling them away from the center of the confinement domain (Mercer et al., Reference Mercer, Chen and Thoreson2011a). The lipid constituents of vesicle membranes do not appear to differ from plasma membrane lipids (Takamori et al., Reference Takamori, Holt, Stenius, Lemke, Grønborg, Riedel, Urlaub, Schenck, Brügger, Ringler, Müller, Rammner, Gräter, Hub, De Groot, Mieskes, Moriyama, Klingauf, Grubmüller, Heuser, Wieland and Jahn2006), suggesting that this increased channel movement is probably not due to changes in local lipid composition but more likely due to a brief expansion of the active zone membrane. Channels rebound towards the center of the confinement domain soon after the termination of exocytosis with a time course similar to that of endocytotic membrane retrieval. Thus, changes in the rate of vesicle fusion may cause the synaptic membrane to inflate and deflate like a balloon. These results are consistent with the suggestion that the fusion of synaptic vesicles may disorganize the arrangement of nearby exocytotic proteins and thereby contribute to postsynaptic depression (Neher & Sakaba, Reference Neher and Sakaba2008; Hosoi et al., Reference Hosoi, Holt and Sakaba2009; Kim & von Gersdorff, Reference Kim and von Gersdorff2009). Disorganization of the synapse by altered membrane fusion and retrieval may also explain the presence of malformed ribbons produced by mutations of proteins involved in exocytosis and endocytosis.

Disruption of the actin cytoskeleton or depletion of cholesterol from cholesterol-rich lipid rafts at the base of the ribbon expand the size of the Ca_V channel confinement domain, allowing channels to move further away from the ribbon (Mercer et al., Reference Mercer, Chen and Thoreson2011a). Ca²⁺ channels are normally confined to a region within 50–100 nm of release sites (Mercer et al., Reference Mercer, Rabl, Riccardi, Brecha, Stella and Thoreson2011b). Increasing that distance would be expected to reduce the probability that an individual channel opening could induce vesicle fusion and thereby diminish the coupling efficiency between channel opening and vesicle fusion. Changes in postsynaptic receptor mobility can produce changes in synaptic strength (Heine et al., Reference Heine, Groc, Frischknecht, Béïque, Lounis, Rumbaugh, Huganir, Cognet and Choquet2008; Petrini et al., Reference Petrini, Lu, Cognet, Lounis, Ehlers and Choquet2009; Rust et al., Reference Rust, Gurniak, Renner, Vara, Morando, Görlich, Sassoè-Pognetto, Banchaabouchi, Giustetto, Triller, Choquet and Witke2010). For example, increasing the lateral mobility of AMPA receptors in the hippocampus promotes recovery from desensitization by speeding the return of undesensitized receptors to the synapse (Heine et al., Reference Heine, Groc, Frischknecht, Béïque, Lounis, Rumbaugh, Huganir, Cognet and Choquet2008). Turnover of actin promoted by n-cofilin enhances AMPA receptor mobility and impairs both long-term potentiation and long-term depression (Rust et al., Reference Rust, Gurniak, Renner, Vara, Morando, Görlich, Sassoè-Pognetto, Banchaabouchi, Giustetto, Triller, Choquet and Witke2010). By analogy, we hypothesize that changes in Ca_V channel mobility caused by changes in endocytotic activity, actin, cholesterol-rich lipid rafts, or other partner proteins might also be a mechanism for regulating release presynaptically.