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.