Yu S P, O'Malley D M, Adams P R
Howard Hughes Medical Institute, Department of Neurobiology and Behavior, State University of New York at Stony Brook 11794-5230.
J Neurosci. 1994 Jun;14(6):3487-99. doi: 10.1523/JNEUROSCI.14-06-03487.1994.
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.
利用全细胞记录、细胞内灌注和共聚焦钙成像技术，在离体牛蛙交感神经节B细胞中研究了细胞内游离钙对M电流（lM）的调节作用。将BAPTA（20 mM）和适量的钙添加到移液管溶液中，以将钙钳制在不同水平。高浓度的BAPTA本身对lM有轻微抑制作用。细胞内灌注有效地控制了细胞内游离钙；用钙指示剂fluo-3进行共聚焦成像证实了这一点。在无钙环境（细胞膜两侧均未添加钙）中，平均lM为166 pA。将细胞内游离钙提高到60 nM或更高可使lM可逆性增强100%以上。当钙从0提高到120 nM时，最大M电导增加一倍，并伴有半激活电压-11 mV的偏移。提高钙浓度也改变了lM关闭和重新开放弛豫的动力学。钙对lM的增强作用需要移液管中有ATP。TEA（5 mM）和d-筒箭毒碱（d-TC；100 microM）不改变钙的作用，表明被调节的是M电流而不是其他K+电流。高钙（450 nM）降低lM。lM的上调和下调与通过钙成像观察到的荧光强度的增加和减少平行。改变细胞外钙对lM或细胞荧光没有显著影响。还探讨了钙在毒蕈碱和肽能对lM调节中的作用。毒蕈碱（1或10 microM）在零钙时对lM的抑制作用小于在较高钙时。在20 mM BAPTA存在下，120 nM钙时几乎完全抑制。在120 nM钙时洗脱毒蕈碱后lM过度恢复，而在零钙时lM几乎没有过度恢复。当使用肽LHRH抑制lM时，在零钙和120 nM钙时观察到类似的效果。我们得出结论，游离钙的绝对水平决定了lM的大小，并且毒蕈碱对lM的最佳抑制和过度恢复都需要最低持续钙水平。虽然我们的数据表明静息钙水平在毒蕈碱抑制中起允许作用，但不能排除激动剂诱导的钙增加的额外作用。
