ON THE MECHANISM OF M-CURRENT INHIBITION BY MUSCARINIC M1 RECEPTORS IN DNA-TRANSFECTED RODENT NEUROBLASTOMA X GLIOMA-CELLS

1. Acetylcholine (ACh) produces two membrane current changes when applied to NG108-15 mouse neuroblastoma x rat glioma hybrid cells transformed (by DNA transfection) to express m1 muscarinic receptors: it activates a Ca2+-dependent K+ conductance, producing an outward current, and it inhibits a voltage-dependent K+ conductance (the M conductance), thus diminishing the M-type voltage-dependent K+ current (I(K(M))) and producing an inward current. The present experiments were undertaken to find out how far inhibition of I(K(M)) might be secondary to stimulation of phospholipase C, by recording membrane currents and intracellular Ca2+ changes with indo-1 using whole-cell patch-clamp methods. 2. Bath application of 100 muM ACh reversibly inhibited I(K(M)) by 47.3+/-3.2% (n = 23). Following pressure-application of 1 mM ACh, the mean latency to inhibition was 420 ms at 35-degrees-C and 1.79 s at 23-degrees-C. Latencies to inhibition by Ba2+ ions were 148 ms at 35-degrees-C and 92 ms at 23-degrees-C. 3. The involvement of a G-protein was tested by adding 0.5 mM GTP-gamma-S or 10 mM potassium fluoride to the pipette solution. These slowly reduced I(K(M)), with half-times of about 30 and 20 min respectively, and rendered the effect of superimposed ACh irreversible. Effects of ACh were not significantly changed after pretreatment for 24 h with 500 ng ml-1 pertussis toxin or on adding up to 10 mM GDP-beta-S to the pipette solution. 4. The role of phospholipase C and its products was tested using neomycin (to inhibit phospholipase C), inositol 1,4,5-trisphosphate (InsP3) and inositol 1,3,4,5-tetrakisphosphate (InaP4), heparin, and phorbol dibutyrate (PDBu) and staurosporin (to activate and inhibit protein kinase C respectively). Both neomycin (1 mM external) and InsP3 (100 muM intrapipette) inhibited the ACh-induced outward current and/or intracellular Ca2+ transient but did not block ACh-induced inhibition of I(K(M)). Intrapipette heparin (1 mM) blocked activation of I(K(Ca)) and reduced Ach-induced inhibitions of I(K(M)), but also reduced inhibition of I(Ca) via endogenous m4 receptors. PDBu (with or without intrapipette ATP) and staurosporin had no significant effects. 5. ACh induced a transient rise in intracellular [Ca2+] but this did not appear to be responsible for inhibition of I(K(M)) since (a) the latter preceded the rise in [Ca2+] by 3.6 s, and (b) ACh still inhibited I(K(M)) when the rise in [Ca2+] was suppressed by (i) repetitive ACh applications, (ii) addition of 100 muM InsP3 to the pipette solution, and (iii) buffering with 20 mM BAPTA. All three procedures inhibited the ACh-induced outward current. 6. Inhibitors of phospholipase A2, lipoxygenase, cyclo-oxygenase or nitric oxide synthase had no significant effect on ACh-induced inhibition of I(K(M)). 7. The presence of GTP (2 mM), ATP (2 mM), dibutyryl-cAMP (1 mM), ATP-gamma-S, (500 muM), adenylyl-imidodiphosphate (AMP-PNP, 2 mM), calmodulin (10 muM), calmodulin antipeptide (10 muM) or the growth-associated protein, GAP-43 (2.5 mM) in the pipette did not affect M-current or modify responses to acetylcholine. 8. It is concluded that ACh-induced inhibition of I(K(M)) is unlikely to be mediated by products of phospholipase C stimulation, either individually or in combination. It is suggested that activation of phospholipase C and inhibition of I(K(M)) represent parallel pathway responses to ACh, and that I(K(M)) might be inhibited by a direct effect of the activated G-protein(s) or through another (unidentified) enzymatic product.