Involvement of Ca 2+ in Signaling Mechanisms Mediating Muscarinic Inhibition of M Currents in Sympathetic Neurons.

Acetylcholine can excite neurons by suppressing M-type (KCNQ) potassium channels. This effect is mediated by M 1 muscarinic receptors coupled to the G q protein. Although PIP 2 depletion and PKC activation have been strongly suggested to contribute to muscarinic inhibition of M currents (I M ), direct evidence is lacking. We investigated the mechanism involved in muscarinic inhibition of I M with Ca 2+ measurement and electrophysiological studies in both neuronal (rat sympathetic neurons) and heterologous (HEK cells expressing KCNQ2/KCNQ3) preparations. We found that muscarinic inhibition of I M was not blocked either by PIP 2 or by calphostin C, a PKC inhibitor. We then examined whether muscarinic inhibition of I M uses multiple signaling pathways by blocking both PIP 2 depletion and PKC activation. This maneuver, however, did not block muscarinic inhibition of I M . Additionally, muscarinic inhibition of I M was not prevented either by sequestering of G-protein βγ subunits from G α -transducin or anti-G βγ antibody or by preventing intracellular trafficking of channel proteins with blebbistatin, a class-II myosin inhibitor. Finally, we re-examined the role of Ca 2+ signals in muscarinic inhibition of I M . Ca 2+ measurements showed that muscarinic stimulation increased intracellular Ca 2+ and was comparable to the Ca 2+ mobilizing effect of bradykinin. Accordingly, 20-mM of BAPTA significantly suppressed muscarinic inhibition of I M . In contrast, muscarinic inhibition of I M was completely insensitive to 20-mM EGTA. Taken together, these data suggest a role of Ca 2+ signaling in muscarinic modulation of I M . The differential effects of EGTA and BAPTA imply that Ca 2+ microdomains or spatially local Ca 2+ signals contribute to inhibition of I M .