Modulation of Ca²⁺ channels by PTX-sensitive G-proteins is blocked by N- ethylmaleimide in rat sympathetic neurons

The actions of N-ethylmaleimide (NEM), a sulfhydryl alkylating agent, on G-protein-mediated inhibition of N-type Ca²⁺ channels in adult rat superior cervical ganglion (SCG) neurons were studied using whole-cell voltage clamp. In SCG neurons, inhibition of I(Ca) occurs by at least three separable pathways: one pertussis toxin (PTX) sensitive and voltage dependent, and two PTX insensitive and voltage independent. NEM blocked PTX-sensitive inhibition nearly completely, with only small effects on PTX-insensitive inhibition. Somatostatin inhibition is completely PTX sensitive and was wholly blocked by a 120 sec exposure to 50 μM NEM, with shorter exposure times producing a less complete block. Inhibition of I(Ca) by norepinephrine (NE) is approximately half PTX sensitive and was also approximately half NEM sensitive. One component of muscarinic inhibition is PTX insensitive, voltage independent, and mediated by a diffusible cytoplasmic messenger; this pathway was largely spared by NEM treatment. Another pathway is also PTX insensitive and voltage independent, used by substance P, and was also largely NEM insensitive. Hence, in SCG neurons, NEM selectively inactivates PTX-sensitive G-proteins. We also find evidence that the PTX-insensitive action of NE is distinct from the other PTX-insensitive pathways, and therefore assign it to a fourth signaling pathway.