Retinoschisin, a New Binding Partner for L-type Voltage-gated Calcium Channels in the Retina

The L-type voltage-gated calcium channels (L-VGCCs) are activated under high depolarization voltages. They are vital for diverse biological events, including cell excitability, differentiation, and synaptic transmission. In retinal photoreceptors, L-VGCCs are responsible for neurotransmitter release and are under circadian influences. However, the mechanism of L-VGCC regulation in photoreceptors is not fully understood. Here, we show that retinoschisin, a highly conserved extracellular protein, interacts with the L-VGCCα1D subunit and regulates its activities in a circadian manner. Mutations in the gene encoding retinoschisin (RS1) cause retinal disorganization that leads to early onset of macular degeneration. Since ion channel activities can be modulated through interactions with extracellular proteins, disruption of these interactions can alter physiology and be the root cause of disease states. Co-immunoprecipitation and mammalian two-hybrid assays showed that retinoschisin and the N-terminal fragment of the L-VGCCα1 subunit physically interacted with one another. The expression and secretion of retinoschisin are under circadian regulation with a peak at night and nadir during the day. Inhibition of L-type VGCCs decreased membrane-bound retinoschisin at night. Overexpression of a missense RS1 mutant gene, R141G, into chicken cone photoreceptors caused a decrease of L-type VGCC currents at night. Our findings demonstrate a novel bidirectional relationship between an ion channel and an extracellular protein; L-type VGCCs regulate the circadian rhythm of retinoschisin secretion, whereas secreted retinoschisin feeds back to regulate L-type VGCCs. Therefore, physical interactions between L-VGCCα1 subunits and retinoschisin play an important role in the membrane retention of L-VGCCα1 subunits and photoreceptor-bipolar synaptic transmission.Interactions between ion channels and extracellular proteins play important roles in the modulation of ion channel gating and synaptic plasticity. For example, extracellular matrix and cell adhesion proteins, such as integrin, regulate L-type voltagegated calcium channel (VGCC)³ functions in vascular smooth muscles (1-3). Likewise, in mice deficient for the extracellular matrix glycoprotein tenascin-C, the L-type VGCC-dependent form of synaptic plasticity in the hippocampus is impaired (4). In vertebrate retinas, retinoschisin is an extracellular adhesion protein secreted primarily from photoreceptors and bipolar cells and is distributed throughout the retina (5-8). After secretion, retinoschisin localizes mainly to the surface of photoreceptors and bipolar cells in adults (9). Mutations in retinoschisin (RS1) cause X-linked retinoschisis (XLRS), a retinal dystrophy that features disorganization of retinal cell layers, disruption of the synaptic structures and neurotransmission between photoreceptors and bipolar cells, and progressive degeneration of rod and cone photoreceptor cells (7, 10-14). Hence, retinoschisin is believed to play an important role in the development and maintenance of retinal cytoarchitecture (7, 8, 15). An interesting clinical aspect of XLRS is that it shares several quantifiable features with X-linked incomplete congenital stationary night blindness (XLCSNB), which is an inherited retinal dystrophy with a mutation in the L-type VGCCα1 subunit gene (16, 17). The electroretinogram (ERG) recordings from XLRS and XLCSNB patients are comparable, and in both cases, the cone photoreceptor responses are more severely affected than the rod responses, and synaptic transmission between photoreceptors and bipolar cells is severely damaged (18). Therefore, we postulated that molecular interactions between L-type VGCCs and retinoschisin may have an important impact in the synaptic transmission between photoreceptors and bipolar cells.Visual systems must be able to detect images despite large daily changes in ambient illumination between day and night. Biological clocks (circadian oscillators) in the retina provide a mechanism that allows the visual system to anticipate these daily changes in photon flux by modulating retinal structure and physiology (19, 20). Retina photoreceptors are nonspiking neurons, and the continuous release of glutamate in the dark is a result of depolarization-evoked activation of L-type VGCCs (21). In avian retina photoreceptors, the circadian rhythms in the synthesis and release of melatonin are also L-type VGCC-dependent (22, 23). There is a diurnal rhythm of L-type VGCC currents in the goldfish retina with average peak amplitudes significantly larger at midnight than at midday (24). In chick retinas, expression of L-type VGCCα1 subunit transcripts and proteins is under circadian control, and the current amplitudes and the VGCCα1 subunit expression in cone photoreceptors are higher during the subjective night than during the subjective day (25). Previously, we showed that retinoschisin mRNA and protein expression and secretion are also under circadian control (26). Intriguingly, the rhythmic expression and secretion of retinoschisin is coincident with the circadian regulation of L-VGCCs in chick photoreceptors (25, 26), and the circadian-regulated secretion of retinoschisin is an L-type VGCC-dependent process; inhibition of L-type VGCCs dampens the circadian rhythm of retinoschisin secretion, where only night time secretion is affected (26). These observations led us to investigate the molecular and functional interactions between L-VGCCα1 subunits and retinoschisin and their functional aspects.In this study, we present the first reported evidence for a novel reciprocal relationship between an ion channel and an extracellular protein; L-type VGCCs govern the circadian rhythm of retinoschisin secretion, whereas secreted retinoschisin ensures the circadian regulation of L-type VGCCs in the retina photoreceptors. The physical interactions between VGCCα1D N-terminal fragment and retinoschisin underlie a positive feedback regulation on each other. Since the N-terminal fragments among L-type VGCCα1C, -1D, and -1F are highly conserved, retinoschisin may very well interact with all three VGCCα1 subunits. Hence, physical interactions between L-VGCCα1 subunits and retinoschisin may play an important role in the membrane retention of VGCCα1 subunits and photoreceptor-bipolar synaptic transmission. Interactions between ion channels and extracellular proteins not only modulate ion channel activities, they may also dictate the physiology and function of cells, and the loss of such interactions may contribute to the cause of a disease state. We also cloned the full-length retinoschisin gene rs1 from chickens (Gallus gallus) and found that retinoschisin is highly conserved among chickens, mice, and humans, and therefore, retinoschisin may be very important in retina function and physiology across species.