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-l 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 approximately 500 at 100 nM, decreasing to approximately 250 at [Ca2+]i approximately 1 pM, 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 allkalinisation (pH 9) or intracellular carboxyeosin both increased resting [Ca2+]i and prolonged the recovery of Ca2+ transients at peak [Ca2+]i C 500 nM. For [Ca2+]i loads >500 nM, recovery showed an additional plateau phase that was abolished i nm-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.
Wanaverbecq
N
N
Department of Pharmacology, University College London, UK. n.wanaverbecq@ion.ucl.ac.uk
Marsh
S J
SJ
Al-Qatari
M
M
Brown
D A
DA
D000818:Animals; D002021:Buffers; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism; D027682:Cation Transport Proteins; D002478:Cells, Cultured; D004594:Electrophysiology; D006706:Homeostasis; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000378:metabolism*; D008928:Mitochondria / Q000502:physiology; D008959:Models, Neurological; D009474:Neurons / Q000378:metabolism*; D009994:Osmolar Concentration; D018408:Patch-Clamp Techniques; D053499:Plasma Membrane Calcium-Transporting ATPases; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D019831:Sodium-Calcium Exchanger / Q000502:physiology; D017783:Superior Cervical Ganglion / Q000166:cytology / Q000378:metabolism*; D013997:Time Factors
D002021:Buffers; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D053499:Plasma Membrane Calcium-Transporting ATPases; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium