Muscarine modulates Ca2+ channel currents in rat sensorimotor pyramidal cells via two distinct pathways

We used the whole cell patch-clamp technique and single-cell reverse transc ription-polymerase chain reaction (RT-PCR) to study the muscarinic receptor -mediated modulation of calcium channel currents in both acutely isolated a nd cultured pyramidal neurons from rat sensorimotor cortex. Single-cell RT- PCR profiling for muscarinic receptor mRNAs revealed the expression of m1, m2, m3, and m4 subtypes in these cells. Muscarine reversibly reduced Ca2+ c urrents in a dose-dependent manner. The modulation was blocked by the musca rinic antagonist atropine. When the internal recording solution included 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid ( EGTA) or 10 mM bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), the modulation was rapid (tau(onset) similar to 1.2 s). Under conditions where intracellular calcium levels were less controlled (0.0-0.1 mM BAPTA), a sl owly developing component of the modulation also was observed (tau(onset) s imilar to 17 s). Both fast and slow components also were observed in record ings with 10 mM EGTA or 20 mM BAPTA when Ca2+ was added to elevate internal [Ca2+] (similar to 150 nM). The fast component was due to a reduction in b oth N- and P-type calcium currents, whereas the slow component involved L-t ype current. N-ethylmaleimide blocked the fast component but not the slow c omponent of the modulation. Preincubation of cultured neurons with pertussi s toxin (PTX) also greatly reduced the fast portion of the modulation. Thes e results suggest a role for both PTX-sensitive G proteins as well as PTX-i nsensitive G proteins in the muscarinic modulation. The fast component of t he modulation was reversed by strong depolarization, whereas the slow compo nent was not. Reblock of the calcium channels by G proteins (at -90 mV) occ urred with a median tau of 68 ms. We conclude that activation of muscarinic receptors results in modulation of N- and P-type channels by a rapid, volt age-dependent pathway and of L-type current by a slow, voltage-independent pathway.