Assignment of muscarinic receptor subtypes mediating G-protein modulation of Ca2+ channels by using knockout mice

There are five known subtypes of muscarinic receptors (M1–M5). We have used knockout mice lacking the M1, M2, or M4 receptors to determine which subtypes mediate modulation of voltage-gated Ca2+ channels in mouse sympathetic neurons. Muscarinic agonists modulate N- and L-type Ca2+ channels in these neurons through two distinct G-protein-mediated mechanisms. One pathway is fast and membrane-delimited and inhibits N- and P/Q-type channels by shifting their activation to more depolarized potentials. The other is slow and voltage-independent and uses a diffusible cytoplasmic messenger to inhibit both Ca2+ channel types. Using patch-clamp methods on acutely dissociated sympathetic neurons, we isolated each pathway by pharmacological and kinetic means and found that each one is nearly absent in a particular knockout mouse. The fast and voltage-dependent pathway is lacking in the M2 receptor knockout mice; the slow and voltage-independent pathway is absent from the M1 receptor knockout mice; and neither pathway is affected in the M4 receptor knockout mice. The knockout effects are clean and are apparently not accompanied by compensatory changes in other muscarinic receptors