Abstract
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).