REGULATION OF M-CURRENT BY INTRACELLULAR CALCIUM IN BULLFROG SYMPATHETIC-GANGLION NEURONS

Regulation of M current (I-M) by intracellular free calcium was studie d in dissociated bullfrog sympathetic ganglion B cells using whole-cel l recording, intracellular perfusion, and confocal calcium imaging. BA PTA (20 mM) and appropriate amounts of calcium were added to pipette s olutions to clamp calcium at different levels. A high concentration of BAPTA itself mildly inhibited I-M. Intracellular perfusion effectivel y controlled cellular free calcium; this was confirmed by confocal ima ging with the calcium indicator fluo-3. In a calcium-free environment (no calcium added to either side of the cell membrane), average I-M wa s 166 pA. Raising intracellular free calcium to 60 nM or higher revers ibly enhanced I-M by more than 100%. The maximum M conductance doubled upon raising calcium from 0 to 120 nM, and was accompanied by a - 11 mV shift of the half-activation voltage. The kinetics of the closing a nd reopening relaxations of I-M were also altered by raising calcium. Enhancement of I-M by calcium required ATP in the pipette. TEA (5 mM) and d-tubocurarine (d-TC; 100 mu M) did not alter the calcium effect, indicating that it was the M current being modulated and not other Kcurrents. High calcium (450 nM) reduced I-M. The up- and downregulatio n of I-M paralleled the increases and decreases of fluorescence intens ity observed via calcium imaging. Changing extracellular calcium had n o significant effect on I-M or cellular fluorescence. The role of calc ium in muscarinic and peptidergic modulation of I-M was also explored. Muscarine (1 or 10 mu M) inhibited I-M less at zero calcium than at h igher calcium. Nearly complete suppression occurred with 120 nM calciu m in the presence of 20 mM BAPTA. I-M overrecovered upon washout of mu scarine at 120 nM calcium, while little overrecovery of I-M developed at zero calcium. Similar effects were observed at zero and 120 nM calc ium when using the peptide LHRH to inhibit I-M. We conclude that the a bsolute level of free calcium determines the size of I-M, and that a m inimum sustained level of calcium is required both for optimal suppres sion of I-M by muscarine and for overrecovery. While our data