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.

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 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 and buffering of cytosolic Ca both inhibit the response to NO. In Ca imaging experiments, we confirm that NO increases cytosolic Ca in amacrine cell processes by activating a Ca influx pathway. Neither the increase in sPSC frequency nor the cytosolic Ca elevations are dependent upon Ca release from stores. NO also enhances evoked GABAergic responses. Because voltage-gated Ca channel function is not altered by NO, the increased evoked response is likely due to the combined effect of voltage-dependent Ca influx adding to the NO-dependent, voltage-independent, Ca influx. Insight into the identity of the Ca 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 elevations. These data suggest that NO production in the inner retina will enhance Ca-dependent GABA release from amacrine cells by activating TRPC channel(s). 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 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.

γ-Amino butyric acid (GABA) and glycine typically mediate synaptic inhibition because their ligand-gated ion channels support the influx of Cl However, the electrochemical gradient for Cl across the postsynaptic plasma membrane determines the voltage response of the postsynaptic cell. Typically, low cytosolic Cl levels support inhibition, whereas higher levels of cytosolic Cl can suppress inhibition or promote depolarization. We previously reported that nitric oxide (NO) releases Cl from acidic organelles and transiently elevates cytosolic Cl, making the response to GABA and glycine excitatory. In this study, we test the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the NO-dependent efflux of organellar Cl We first establish the mRNA and protein expression of CFTR in our model system, cultured chick retinal amacrine cells. Using whole cell voltage-clamp recordings of currents through GABA-gated Cl channels, we examine the effects of pharmacological inhibition of CFTR on the NO-dependent release of internal Cl To interfere with the expression of CFTR, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. We find that both pharmacological inhibition and CRISPR/Cas9-mediated knockdown of CFTR block the ability of NO to release Cl from internal stores. These results demonstrate that CFTR is required for the NO-dependent efflux of Cl from acidic organelles. Although CFTR function has been studied extensively in the context of epithelia, relatively little is known about its function in neurons. We show that CFTR is involved in an NO-dependent release of Cl from acidic organelles. This internal function of CFTR is particularly relevant to neuronal physiology because postsynaptic cytosolic Cl levels determine the outcome of GABA- and glycinergic synaptic signaling. Thus the CFTR may play a role in regulating synaptic transmission.

Voltage-gated Cav1 and Cav2 Ca2+ channels are comprised of a pore-forming a1 subunit (Cav1.1-1.4, Cav2.1-2.3) and auxiliary β (β1-4) and α2δ (α2δ-1-4) 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 (Cav1.4+ex47, Cav1.4Δex47, respectively), and the auxiliary subunits, β2x13 and α2δ-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 β2x13 and α2δ-4 but not in those with β2A and α2δ-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 β2x13 and α2δ-4. Our results reveal complex actions of IT in modifying the properties of Cav1.4 channels, which may influence the pathological consequences of this mutation in retinal photoreceptors.

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 channels in cultured retinal ACs. This voltage-independent quantal GABA release relies on a Ca 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 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, 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 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 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.