title: 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 (I(ClCa)) in protocols that manipulated Ca2+-buffering. I(ClCa)was activated by Ca2+ influx via voltage-gated Ca2+ channels or by Ca2+ release via RyRs activated by 10 mM caffeine. I(ClCa)was measured in neurons at 20 degrees C and 35 degrees C using the amphotericin perforated patch technique that preserves endogenous Ca2+-buffering, or at 20 degrees C in neurons dialyzed with pipette solutions designed to replace the endogenous Ca2+ buffers. The amplitude of I(ClCa)activated by Ca2+ influx or Ca2+ at 20 degrees C was similar in the amphotericin neurons and neurons dialyzed with an 'unbuffered' pipette solution containing 10 mM citrate and 3 mM ATP as the only Ca2+ binding molecules. Thus, endogenous mobile Ca2+ buffers are relatively unimportant in chick DRG neurons. Warming the neurons from 20 degrees C to 35 degrees C increased the amplitude and the rate of deactivation of I(ClCa)consistent with an increased rate of Ca2+ buffering by fixed endogenous Ca2+-buffers. Dialysis with 2 mM EGTA/0.1 microM free Ca2+ reduced the amplitude and increased the rate of deactivation of I(ClCa)activated by Ca2+ influx and abolished I(ClCa)activated by Ca2+ release. Dialysis with 2 mM BAPTA/0.1 microM free Ca2+ abolished I(ClCa)activated by Ca2+ influx or release. Dialysis with 42 mM HEEDTA/0.5 microM free Ca2+ caused the persistent activation of I(ClCa). Calculations using a Ca2+-diffusion model suggest that the voltage-gated Ca2+ channels and the Ca2+-activated Cl- channels are separated by 50-400 nm and that the RyRs are more than 600 nm from the plasma membrane.
authors.affiliation: Department of Physiology & Cell Biology/MS 352, University of Nevada School of Medicine, Reno, NV, 89557, USA.
authors.forename: S M
authors.initials: SM
authors.lastname: Ward
authors.forename: J L
authors.initials: JL
authors.lastname: Kenyon
mesh_terms: D000666:Amphotericin B / Q000494:pharmacology; D000818:Animals; D002021:Buffers; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002642:Chick Embryo; D018118:Chloride Channels / Q000502:physiology; D003593:Cytoplasm / Q000378:metabolism; D003956:Dialysis; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005727:Ganglia, Spinal / Q000166:cytology; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D008854:Microscopy, Electron; D009475:Neurons, Afferent / Q000378:metabolism* / Q000648:ultrastructure; D018408:Patch-Clamp Techniques; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*
chemical_list: D002021:Buffers; D015220:Calcium Channels; D018118:Chloride Channels; D019837:Ryanodine Receptor Calcium Release Channel; D004533:Egtazic Acid; D000666:Amphotericin B; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium