Selective disruption by protein kinases of G-protein-mediated Ca²⁺ channel modulation

1. We studied the effects of phorbol-12-myristate, 13-acetate (PMA) on G-protein-mediated inhibition of Ca²⁺ channels by several neurotransmitters in rat superior cervical ganglion (SCG) sympathetic neurons, with the use of the whole cell patch clamp. PMA attenuated membrane-delimited inhibition of calcium currents (I(Ca)) by norepinephrine (NE) and somatostatin by more than half, but did not attenuate inhibition by M₁ muscarinic receptors, which use a diffusible cytoplasmic messenger. Inhibition of I(Ca) by NE through pertussis-toxin-sensitive and -insensitive G proteins was equally attenuated by PMA. PMA enhanced I(Ca) in about half the neurons (enhancement of 10 ± 1%, mean ± SE) and strongly reduced the holding current in 44 of 61 cells. 2. The M-type K⁺ current (I(M)) was not suppressed by PMA, and PMA did not attenuate inhibition of I(M) by muscarinic agonists, which is also via a diffusible cytoplasmic messenger. 3. Attenuation of NE and somatostatin inhibition by PMA was blocked by 1 μM staurosporine, a broad-spectrum protein kinase inhibitor. Tests with three inhibitors selective for distinct isoforms of protein kinase C (PKC) gave mixed results. PMA's actions were unaffected by 1 μM calphostin C, blocked by 500 nM bisindolylmaleimide, and unaffected by the pseudosubstrate inhibitor PKC_19-36. 4. Thus we find that two membrane-delimited signaling pathways that inhibit ion channels in rat SCG neurons are strongly attenuated by PMA, but signaling pathway(s) that use a diffusible cytoplasmic messenger are not. We speculate that a nonstandard PKC isoform, perhaps PKCμ, mediates PMA actions.