Involvement of Ca2+ in Signaling Mechanisms Mediating Muscarinic Inhibition of M Currents in Sympathetic Neurons

Abstract

Acetylcholine can excite neurons by suppressing M-type (KCNQ) potassium channels. This effect is mediated by M₁ muscarinic receptors coupled to the G_q protein. Although PIP₂ 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 Ca²⁺ 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 PIP₂ or by calphostin C, a PKC inhibitor. We then examined whether muscarinic inhibition of I_M uses multiple signaling pathways by blocking both PIP₂ 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 Ca²⁺ signals in muscarinic inhibition of I_M. Ca²⁺ measurements showed that muscarinic stimulation increased intracellular Ca²⁺ and was comparable to the Ca²⁺ 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 Ca²⁺ signaling in muscarinic modulation of I_M. The differential effects of EGTA and BAPTA imply that Ca²⁺ microdomains or spatially local Ca²⁺ signals contribute to inhibition of I_M.

Keywords: Ca2+; KCNQ potassium channel; Muscarinic receptor; PIP2; PKC; Sympathetic neurons.