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Journal of Neuroscience, Vol 16, 1008-1015, Copyright © 1996 by Society for Neuroscience
Modulation of calcium efflux from cultured rat dorsal root ganglion neurons
JL Werth, YM Usachev and SA Thayer
Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455, USA.
The free intracellular Ca2+ concentration ([Ca2+]i) is governed by the
balance between the activation of Ca2+ channels and buffering and efflux
processes. We tested the hypothesis that Ca2+ efflux pathways are
susceptible to modulation. The whole-cell patch-clamp technique was used in
combination with Indo-1-based microfluorometry to record Ca2+ current and
[Ca2+]i simultaneously from single rat dorsal root ganglion (DRG) neurons
grown in culture. Depolarizing test pulses (-80 to 0 mV, 100-300 msec)
elicited [Ca2+]i transients that recovered to basal levels by a process
best-fit with a single exponential (tau = 5.1 +/- 0.4 sec; n = 14) and were
independent of Ca2+ load (40-500 pC) over this range of test pulses.
[Ca2+]i transients recorded in whole-cell configuration were similar to
those elicited by a brief train of action potentials in unclamped neurons.
Inhibition of Ca2+ sequestration into intracellular stores with
thapsigargin had no effect on the kinetics of recovery. Inhibition of
plasma membrane Ca2+ ATPase (PMCA) function by including a peptide
inhibitor (C28R2) in the patch pipette significantly slowed recovery to
basal [Ca2+]i (tau = 9.9 +/- 0.8 sec; n = 4). Preincubation with
calmidazolium, a calmodulin antagonist, produced modest slowing of Ca2+
efflux. Phorbol dibutyrate, an activator of protein kinase C (PKC),
accelerated Ca2+ efflux only when the PMCA had been inhibited by C28R2. We
conclude that in DRG neurons PMCAs are responsible for lowering [Ca2+]i
after small Ca2+ loads and that PMCA-mediated Ca2+ efflux is modulated by
calmodulin- and PKC- signaling pathways.
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