Modulation of calcium efflux from cultured rat dorsal root ganglion neurons

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 (τ = 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) (τ = 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.