Proton-mediated inhibition of L-type Ca²⁺ currents in AII amacrine cells

Abstract

Purpose : Protons released during synaptic vesicle exocytosis can transiently block voltage gated Ca²⁺ channels located at ribbon type active zones. ATP-dependent vesicular pump proteins shuttle protons into the lumen of synaptic vesicles. Proton inhibition of Ca²⁺ current (I_Ca) is characterized by a brief “notch” in I_Ca. However, this has only been observed in select glutamatergic synapses such as rod bipolar cells, photoreceptors, and auditory hair cells, all of which have L-type Ca²⁺ channels located near synaptic ribbons. We observed a similar notch in L-type I_Ca recorded from AII amacrine cells (AIIs), which are inhibitory interneurons in the mammalian retina. Unlike previous reports, AIIs are glycinergic with conventional synapses. We hypothesize that this notch is due to a transient inhibition of the Ca²⁺ channel by protons released during exocytosis.

Methods : Retina slices were prepared from C57 mice (2mo) and Brown Norway rats (9-13mo). Whole-cell voltage-clamp experiments were performed on AIIs using a Cs-based internal with 10 mM TEA. Evoked synaptic vesicle exocytosis was measured from time-resolved membrane capacitance (C_m) recordings using the “sine + DC” method. AIIs were held at -80 mV, and given a 100 ms depolarizing test pulse (-10 mV) between sinusoidal voltage commands (30 mV peak-to-peak; 2kHz). Exocytosis was measured as ΔC_m (=C_m jump - baseline C_m), and I_Ca were resolved using offline leak-subtraction.

Results : C_m jumps measured from rat (n=11) and mouse (n=4) AIIs were not significantly different (rats: 63.4 ± 15.9 pF, mouse: 51 ± 12.9 pF), corresponding to ~1500 vesicles. However, I_Ca in rat AIIs were significantly larger, which was observed by measuring the peak I_Ca (rats: -168.7 ± 22.6 pA, mouse: -138.7 ± 26.6 pA) and Ca²⁺ charge transfer (rat: 16.1 ± 3.14 pC, mouse: 9.13 ± 4.17 pC; p=.004). Furthermore, I_Ca traces from mature rats exhibited a transient notch (100.1 ± 26.7 pA) not seen in mouse.

Conclusions : Our results suggest that proton-mediated inhibition of L-type I_Ca may not be limited to ribbon synapses. The I_Ca notch in rat AIIs suggests that the active zone structure is different in mouse. However, we must first confirm that this notch truly is inhibition from vesicular protons and not current from unblocked K⁺ channels or some type of transporter. Brief inhibition of the I_Ca may provide a fast mechanism for regulating exocytosis over a broad range of light stimuli intensities and frequencies.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.