TRPC5 is required for the NO-dependent increase in dendritic Ca²⁺and GABA release from chick retinal amacrine cells

Abstract

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.

Adenylate Cyclase 1 Links Calcium Signaling to CFTR-Dependent Cytosolic Chloride Elevations in Chick Amacrine Cells

“…The higher magnitude of change NO-dependent cytosolic Ca 2+ in zero Ca 2+ external is due to the lower resting cytosolic Ca 2+ level (normal external, 1,808 ± 131.7 AU; n = 45; zero Ca 2+ external, 1,460 ± 94.2 AU; n = 54; p < 0.05, unpaired t -test, Figure 1G ). The decay time constant in normal external was larger than that in zero Ca 2+ external condition, suggesting that the later part of the response was more dependent upon extracellular Ca 2+ , consistent with activation of TRPC5 (Maddox et al, 2018 ; normal external, 213.8 ± 22.9, n = 33 s; zero Ca 2+ external, 45.8 ± 6.2 s, n = 49; p < 0.0001, unpaired t -test, Figure 1H ). Together, these results suggest that NO-bubbled solutions can elicit both Ca 2+ influx across the plasma membrane and release of Ca 2+ from internal stores.…”

“…1G). The decay time constant in normal external was larger than that in zero Ca 2+ external condition, suggesting that the latter part of the response was more dependent upon extracellular Ca 2+ , consistent with activation of TRPC5 (Maddox et al, 2018;normal external, 213.8 ± 22.9 s, n = 33; zero Ca 2+ external, 45.8 ± 6.2 s, n = 49; p < 0.0001, unpaired t-test, Figure 1H). Together, these results suggest that NO-bubbled solutions can elicit both Ca 2+ influx across the plasma membrane and release of Ca 2+ from internal stores.…”

“…We have previously shown that the NO donor SNAP can enhance intracellular Ca 2+ in ACs via activation of transient receptor potential canonical channel 5 (TRPC5; Maddox and Gleason, 2017 ; Maddox et al, 2018 ). To confirm the NO-bubbled external solution also elevates Ca 2+ levels, cells were loaded with Oregon Green BAPTA-1 488-AM (OGB, 2 μM) and fluorescence intensity was monitored over time.…”

Non-Cell-Autonomous Regulation of Optic Nerve Regeneration by Amacrine Cells

“…In chick ACs in vitro, NO transiently reverses GABA-and glycine-gated currents, converting inhibition of ACs into excitation, thereby increasing the firing of these cells and thus enhanced inhibitory drive on their synaptic partners (e.g., RGCs). This NO-induced shift in E Cl− is likely due to release of Cl − from intracellular stores (Hoffpauir et al, 2006;Krishnan and Gleason, 2015;Krishnan et al, 2017;Maddox and Gleason, 2017;Maddox et al, 2018). In addition, NO may drive synaptic glutamate release from BCs without membrane depolarization via a TRPC Ca 2+ influx-mediated pathway, as shown in the chick retina (Maddox et al, 2018), further depolarizing ACs.…”

Inhibition of endocytosis suppresses the nitric oxide-dependent release of Cl- in retinal amacrine cells

“…The involvement of SVs in the NOdrCl is especially relevant to AC function because ACs participate in serial [ 74 ] and reciprocal synapses [ 21 ] such that presynaptic Cl - can affect the sign of nearby incoming GABAergic synapses in a highly localized fashion [ 22 – 24 ]. We have also recently demonstrated that the NO donor SNAP can increase spontaneous and evoked GABA release via presynaptic activation of TRPC5 channels [ 41 , 75 ] providing an additional NO-dependent mechanism for enhancing GABAergic output from ACs.…”