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

There are five known subtypes of muscarinic receptors (M-1-M-5) We have use d knockout mice lacking the M-1, M-2, or M-4 receptors to determine which s ubtypes mediate modulation of voltage-gated Ca2+ channels in mouse sympathe tic neurons. Muscarinic agonists modulate N- and L-type Ca2+ channels in th ese neurons through two distinct G-protein-mediated mechanisms. One pathway is fast and membrane-delimited and inhibits N- and P/Q-type channels by sh ifting their activation to more depolarized potentials. The other is slow a nd voltage-independent and uses a diffusible cytoplasmic messenger to inhib it both Ca2+ channel types. Using patch-clamp methods on acutely dissociate d sympathetic neurons, we isolated each pathway by pharmacological and kine tic means and found that each one is nearly absent in a particular knockout mouse. The fast and voltage-dependent pathway is lacking in the M-2 recept or knockout mice; the slow and voltage-independent pathway is absent from t he M-1 receptor knockout mice; and neither pathway is affected in the Mg re ceptor knockout mice. The knockout effects are clean and are apparently not accompanied by compensatory changes in other muscarinic receptors.