The spatial relationship between Ca2+channels and Ca2+-activated channels and the function of Ca2+-buffering in avian sensory neurons

Abstract

In order to learn about the endogenous Ca2+-buffering in the cytoplasm of chick dorsal root ganglion (DRG) neurons and the distance separating the ryanodine receptor Ca2+release channels (RyRs) from the plasma membrane, we monitored the amplitude and time course of Ca2+-activated Cl–currents (lCl(Ca)) in protocols that manipulated Ca2+-buffering. lCl(Ca)was activated by Ca2+influx via voltage-gated Ca2+channels or by Ca2+release via RyRs activated by 10mM caffeine. lCl(Ca)was measured in neurons at 20°C and 35°C using the amphotericin perforated patch technique that preserves endogenous Ca2+-buffering, or at 20°C in neurons dialyzed with pipette solutions designed to replace the endogenous Ca2+buffers. The amplitude of lCl(Ca)activated by Ca2+influx or Ca2+at 20°C was similar in the amphotericin neurons and neurons dialyzed with an ‘unbuffered’ pipette solution containing 10mM citrate and 3mM ATP as the only Ca2+binding molecules. Thus, endogenous mobile Ca2+buffers are relatively unimportant in chick DRG neurons. Warming the neurons from 20°C to 35°C increased the amplitude and the rate of deactivation of lCl(Ca)consistent with an increased rate of Ca2+buffering by fixed endogenous Ca2+-buffers. Dialysis with 2mM EGTA/0.1μM free Ca2+reduced the amplitude and increased the rate of deactivation of lCl(Ca)activated by Ca2+influx and abolished lCl(Ca)activated by Ca2+release. Dialysis with 2mM BAPTA/0.1μM free Ca2+abolished lCl(Ca)activated by Ca2+influx or release. Dialysis with 42mM HEEDTA/0.5μM free Ca2+caused the persistent activation of lCl(Ca). Calculations using a Ca2+-diffusion model suggest that the voltage-gated Ca2+channels and the Ca2+-activated Cl–channels are separated by 50–400nm and that the RyRs are more than 600nm from the plasma membrane.