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Synapses are fundamental information processing units that rely on voltage-gated Ca2+ (Cav) channels to trigger Ca2+-dependent neurotransmitter release. Cav channels also play Ca2+-independent roles in other biological contexts, but whether they do so in axon terminals is unknown. Here, we addressed this unknown with respect to the requirement for Cav1.4 L-type channels for the formation of rod photoreceptor synapses in the retina. Using a mouse strain expressing a non-conducting mutant form of Cav1.4, we report that the Cav1.4 protein, but not its Ca2+ conductance, is required for the molecular assembly of rod synapses; however, Cav1.4 Ca2+ signals are needed for the appropriate recruitment of postsynaptic partners. Our results support a model in which presynaptic Cav channels serve both as organizers of synaptic building blocks and as sources of Ca2+ ions in building the first synapse of the visual pathway and perhaps more broadly in the nervous system. [ABSTRACT FROM AUTHOR]

Retinal amacrine cells express nitric oxide (NO) synthase and produce NO, making NO available to regulate the function of amacrine cells. Here we test the hypothesis that NO can alter the GABAergic synaptic output of amacrine cells. We investigate this using whole cell voltage clamp recordings and Ca2+ imaging of cultured chick retinal amacrine cells. When recording from amacrine cells receiving synaptic input from other amacrine cells, we find that NO increases GABAergic spontaneous postsynaptic current (sPSC) frequency. This increase in sPSC frequency does not require the canonical NO receptor, soluble guanylate cyclase, or presynaptic action potentials. However, removal of extracellular Ca2+ and buffering of cytosolic Ca2+ both inhibit the response to NO. In Ca2+ imaging experiments, we confirm that NO increases cytosolic Ca2+ in amacrine cell processes by activating a Ca2+ influx pathway. Neither the increase in sPSC frequency nor the cytosolic Ca2+ elevations are dependent upon Ca2+ release from stores. NO also enhances evoked GABAergic responses. Because voltage-gated Ca2+ channel function is not altered by NO, the increased evoked response is likely due to the combined effect of voltage-dependent Ca2+ influx adding to the NO-dependent, voltage-independent, Ca2+ influx. Insight into the identity of the Ca2+ influx pathway is provided by the transient receptor potential canonical (TRPC) channel inhibitor clemizole, which prevents the NO-dependent increase in sPSC frequency and cytosolic Ca2+ elevations. These data suggest that NO production in the inner retina will enhance Ca2+ -dependent GABA release from amacrine cells by activating TRPC channel(s). [ABSTRACT FROM AUTHOR]

Voltage-gated Cav1 andCav2 Ca2+ channels are comprised of a pore-forming a1 subunit (Cav1.1-1.4, Cav2.1-2.3) and auxiliary b (b1-4) and a2d (a2d2124) subunits. The properties of these channels vary with distinct combinations of Cav subunits and alternative splicing of the encoding transcripts. Therefore, the impact of disease-causing mutations affecting these channels may depend on the identities of Cav subunits and splice variants. Here, we analyzed the effects of a congenital stationary night blindness type 2 (CSNB2)-causing mutation, I745T (IT), in Cav1.4 channels typical of those in human retina: Cav1.4 splice variants with or without exon 47 (Cav1.41ex47 and Cav1.4Dex47, respectively), and the auxiliary subunits, ޚ2X13 and a2d-4. We find that IT caused both Cav1.4 splice variants to activate at significantly more negative voltages and with slower deactivation kinetics than the corresponding WT channels. These effects of the IT mutation, along with unexpected alterations in ion selectivity, were generally larger in channels lacking exon 47. The weaker ion selectivity caused by IT led to hyperpolarizing shifts in the reversal potential and large outward currents that were evident in channels containing the auxiliary subunits ޚ2X13 and a2d-4 but not in those with ޚ2A and a2d-1. We conclude that the IT mutation stabilizes channel opening and alters ion selectivity of Cav1.4 in a manner that is strengthened by exclusion of exon 47 and inclusion of b2X13 and a2d-4. Our results reveal complex actions of IT inmodifying the properties of Cav1.4 channels, which may influence the pathological consequences of this mutation in retinal photoreceptors. [ABSTRACT FROM AUTHOR]

In congenital stationary night blindness type 2 (CSNB2)-a disorder involving the Ca v 1.4 (L-type) Ca 2+ channel-visual impairment is mild considering that Ca v 1.4 mediates synaptic release from rod and cone photoreceptors. Here, we addressed this conundrum using a Ca v 1.4 knockout (KO) mouse and a knock-in (G369i KI) mouse expressing a non-conducting Ca v 1.4. Surprisingly, Ca v 3 (T-type) Ca 2+ currents were detected in cones of G369i KI mice and Ca v 1.4 KO mice but not in cones of wild-type mouse, ground squirrel, and macaque retina. Whereas Ca v 1.4 KO mice are blind, G369i KI mice exhibit normal photopic (i.e., cone-mediated) visual behavior. Cone synapses, which fail to form in Ca v 1.4 KO mice, are present, albeit enlarged, and with some errors in postsynaptic wiring in G369i KI mice. While Ca v 1.4 KO mice lack evidence of cone synaptic responses, electrophysiological recordings in G369i KI mice revealed nominal transmission from cones to horizontal cells and bipolar cells. In CSNB2, we propose that Ca v 3 channels maintain cone synaptic output provided that the nonconducting role of Ca v 1.4 in cone synaptogenesis remains intact. Our findings reveal an unexpected form of homeostatic plasticity that relies on a non-canonical role of an ion channel., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.

Voltage-gated Ca v 1 and Ca v 2 Ca 2+ channels are comprised of a pore-forming α 1 subunit (Ca v 1.1-1.4, Ca v 2.1-2.3) and auxiliary β (β 1-4 ) and α 2 δ (α 2 δ-1-4) subunits. The properties of these channels vary with distinct combinations of Ca v subunits and alternative splicing of the encoding transcripts. Therefore, the impact of disease-causing mutations affecting these channels may depend on the identities of Ca v subunits and splice variants. Here, we analyzed the effects of a congenital stationary night blindness type 2 (CSNB2)-causing mutation, I745T (IT), in Ca v 1.4 channels typical of those in human retina: Ca v 1.4 splice variants with or without exon 47 (Ca v 1.4+ex47 and Ca v 1.4Δex47, respectively), and the auxiliary subunits, β 2X13 and α 2 δ-4. We find that IT caused both Ca v 1.4 splice variants to activate at significantly more negative voltages and with slower deactivation kinetics than the corresponding WT channels. These effects of the IT mutation, along with unexpected alterations in ion selectivity, were generally larger in channels lacking exon 47. The weaker ion selectivity caused by IT led to hyperpolarizing shifts in the reversal potential and large outward currents that were evident in channels containing the auxiliary subunits β 2X13 and α 2 δ-4 but not in those with β 2A and α 2 δ-1. We conclude that the IT mutation stabilizes channel opening and alters ion selectivity of Ca v 1.4 in a manner that is strengthened by exclusion of exon 47 and inclusion of β 2X13 and α 2 δ-4. Our results reveal complex actions of IT in modifying the properties of Ca v 1.4 channels, which may influence the pathological consequences of this mutation in retinal photoreceptors., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Williams et al.)

Synapses are fundamental information processing units that rely on voltage-gated Ca 2+ (Ca v ) channels to trigger Ca 2+ -dependent neurotransmitter release. Ca v channels also play Ca 2+ -independent roles in other biological contexts, but whether they do so in axon terminals is unknown. Here, we addressed this unknown with respect to the requirement for Ca v 1.4 L-type channels for the formation of rod photoreceptor synapses in the retina. Using a mouse strain expressing a non-conducting mutant form of Ca v 1.4, we report that the Ca v 1.4 protein, but not its Ca 2+ conductance, is required for the molecular assembly of rod synapses; however, Ca v 1.4 Ca 2+ signals are needed for the appropriate recruitment of postsynaptic partners. Our results support a model in which presynaptic Ca v channels serve both as organizers of synaptic building blocks and as sources of Ca 2+ ions in building the first synapse of the visual pathway and perhaps more broadly in the nervous system., Competing Interests: JM, KR, RY, BW, JH, PD, VK, LD, SB, NA, MH, AL No competing interests declared, (© 2020, Maddox et al.)

GABAergic signaling from amacrine cells (ACs) is a fundamental aspect of visual signal processing in the inner retina. We have previously shown that nitric oxide (NO) can elicit release of GABA independently from activation of voltage-gated Ca 2+ channels in cultured retinal ACs. This voltage-independent quantal GABA release relies on a Ca 2+ influx mechanism with pharmacological characteristics consistent with the involvement of the transient receptor potential canonical (TRPC) channels TRPC4 and/or TRPC5. To determine the identity of these channels, we evaluated the ability of NO to elevate dendritic Ca 2+ and to stimulate GABA release from cultured ACs under conditions known to alter the function of TRPC4 and 5. We found that these effects of NO are phospholipase C dependent, have a biphasic dependence on La 3+ , and are unaffected by moderate concentrations of the TRPC4-selective antagonist ML204. Together, these results suggest that NO promotes GABA release by activating TRPC5 channels in AC dendrites. To confirm a role for TRPC5, we knocked down the expression of TRPC5 using CRISPR/Cas9-mediated gene knockdown and found that both the NO-dependent Ca 2+ elevations and increase in GABA release are dependent on the expression of TRPC5. These results demonstrate a novel NO-dependent mechanism for regulating neurotransmitter output from retinal ACs. NEW & NOTEWORTHY Elucidating the mechanisms regulating GABAergic synaptic transmission in the inner retina is key to understanding the flexibility of retinal ganglion cell output. Here, we demonstrate that nitric oxide (NO) can activate a transient receptor potential canonical 5 (TRPC5)-mediated Ca 2+ influx, which is sufficient to drive vesicular GABA release from retinal amacrine cells. This NO-dependent mechanism can bypass the need for depolarization and may have an important role in processing the visual signal by enhancing retinal amacrine cell GABAergic inhibitory output.

Retinal amacrine cells express nitric oxide (NO) synthase and produce NO, making NO available to regulate the function of amacrine cells. Here we test the hypothesis that NO can alter the GABAergic synaptic output of amacrine cells. We investigate this using whole cell voltage clamp recordings and Ca 2+ imaging of cultured chick retinal amacrine cells. When recording from amacrine cells receiving synaptic input from other amacrine cells, we find that NO increases GABAergic spontaneous postsynaptic current (sPSC) frequency. This increase in sPSC frequency does not require the canonical NO receptor, soluble guanylate cyclase, or presynaptic action potentials. However, removal of extracellular Ca 2+ and buffering of cytosolic Ca 2+ both inhibit the response to NO. In Ca 2+ imaging experiments, we confirm that NO increases cytosolic Ca 2+ in amacrine cell processes by activating a Ca 2+ influx pathway. Neither the increase in sPSC frequency nor the cytosolic Ca 2+ elevations are dependent upon Ca 2+ release from stores. NO also enhances evoked GABAergic responses. Because voltage-gated Ca 2+ channel function is not altered by NO, the increased evoked response is likely due to the combined effect of voltage-dependent Ca 2+ influx adding to the NO-dependent, voltage-independent, Ca 2+ influx. Insight into the identity of the Ca 2+ influx pathway is provided by the transient receptor potential canonical (TRPC) channel inhibitor clemizole, which prevents the NO-dependent increase in sPSC frequency and cytosolic Ca 2+ elevations. These data suggest that NO production in the inner retina will enhance Ca 2+ -dependent GABA release from amacrine cells by activating TRPC channel(s). NEW & NOTEWORTHY Our research provides evidence that nitric oxide (NO) promotes GABAergic output from retinal amacrine cells by activating a likely transient receptor potential canonical-mediated Ca 2+ influx pathway. This NO-dependent mechanism promoting GABA release can be voltage independent, suggesting that, in the retina, local NO production can bypass the formal retinal circuitry and increase local inhibition., (Copyright © 2017 the American Physiological Society.)