Abstract
Regulation of M current (lM) by intracellular free calcium was studied in dissociated bullfrog sympathetic ganglion B cells using whole-cell recording, intracellular perfusion, and confocal calcium imaging. BAPTA (20 mM) and appropriate amounts of calcium were added to pipette solutions to clamp calcium at different levels. A high concentration of BAPTA itself mildly inhibited lM. Intracellular perfusion effectively controlled cellular free calcium; this was confirmed by confocal imaging with the calcium indicator fluo-3. In a calcium-free environment (no calcium added to either side of the cell membrane), average lM was 166 pA. Raising intracellular free calcium to 60 nM or higher reversibly enhanced lM 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 and reopening relaxations of lM were also altered by raising calcium. Enhancement of lM by calcium required ATP in the pipette. TEA (5 mM) and d-tubocurarine (d-TC; 100 microM) did not alter the calcium effect, indicating that it was the M current being modulated and not other K+ currents. High calcium (450 nM) reduced lM. The up- and downregulation of lM paralleled the increases and decreases of fluorescence intensity observed via calcium imaging. Changing extracellular calcium had no significant effect on lM or cellular fluorescence. The role of calcium in muscarinic and peptidergic modulation of lM was also explored. Muscarine (1 or 10 microM) inhibited lM less at zero calcium than at higher calcium. Nearly complete suppression occurred with 120 nM calcium in the presence of 20 mM BAPTA. lM overrecovered upon washout of muscarine at 120 nM calcium, while little overrecovery of lM developed at zero calcium. Similar effects were observed at zero and 120 nM calcium when using the peptide LHRH to inhibit lM. We conclude that the absolute level of free calcium determines the size of lM, and that a minimum sustained level of calcium is required both for optimal suppression of lM by muscarine and for overrecovery. While our data suggest that resting calcium levels play a permissive role in muscarinic suppression, an additional role for agonist-induced calcium increases cannot be ruled out.