The plasma membrane calcium-ATPase as a major mechanism for intracellular calcium regulation in neurones from the rat superior cervical ganglion

Patch-clamp recording combined with indo-1 measurement of free intracellular calcium concentration ([Ca2+]i) was used to determine the homeostatic systems involved in the maintenance of resting [Ca2+]i and in the clearance of Ca2+ transients following activation of voltage-gated Ca2+ channels in neurones cultured from rat superior cervical ganglion (SCG). The Ca2+ binding ratio was estimated to be ∼500 at 100 nM, decreasing to ∼250 at [Ca2+]i≈ 1 μM, and to involve at least two buffering systems with different affinities for Ca2+. Removal of extracellular Ca2+ led to a decrease in [Ca2+]i that was mimicked by the addition of La3+, and was more pronounced after inhibition of the endoplasmic reticulum Ca2+ uptake system (SERCA). Inhibition of the plasma membrane Ca2+ pump (PMCA) by extracellular alkalinisation (pH 9) or intracellular carboxyeosin both increased resting [Ca2+]i and prolonged the recovery of Ca2+ transients at peak [Ca2+]i≤ 500 nM. For [Ca2+]i loads > 500 nM, recovery showed an additional plateau phase that was abolished in m-chlorophenylhydrazone (CCCP) or on omitting intracellular Na+. Inhibition of the plasma membrane Na+ -Ca2+ exchanger (NCX) and of SERCA had a small but significant additional effect on the rate of decay of these larger Ca2+ transients. In conclusion, resting [Ca2+]i is maintained by passive Ca2+ influx and regulated by a large Ca2+ buffering system, Ca2+ extrusion via a PMCA and Ca2+ transport from the intracellular stores. PMCA is also the principal Ca2+ extrusion system at low Ca2+ loads, with additional participation of the NCX and intracellular organelles at high [Ca2+]i