Muscarinic modulation of erg potassium current.
We studied modulation of current in human embryonic kidney tsA-201 cells coexpressing rat erg1 channels with M(1) muscarinic receptors. Maximal current was inhibited 30% during muscarinic receptor stimulation, with a small positive shift of the midpoint of activation. Inhibition was attenuated by coexpression of the regulator of G-protein signalling RGS2 or of a dominant-negative protein, G(q), but not by N-ethylmaleimide or C3 toxin. Overexpression of a constitutively active form of G(q) (but not of G(13) or of G(s)) abolished the erg current. Hence it is likely that G(q/11), and not G(i/o) or G(13), mediates muscarinic inhibition. Muscarinic suppression of erg was attenuated by chelating intracellular Ca(2+) to < 1 nm free Ca(2+) with 20 mm BAPTA in the pipette, but suppression was normal if internal Ca(2+) was strongly clamped to a 129 nm free Ca(2+) level with a BAPTA buffer and this was combined with numerous other measures to prevent intracellular Ca(2+) transients (pentosan polysulphate, preincubation with thapsigargin, and removal of extracellular Ca(2+)). Hence a minimum amount of Ca(2+) was necessary for the inhibition, but a Ca(2+) elevation was not. The ATP analogue AMP-PCP did not prevent inhibition. The protein kinase C (PKC) blockers staurosporine and bisindolylmaleimide I did not prevent inhibition, and the PKC-activating phorbol ester PMA did not mimic it. Neither the tyrosine kinase inhibitor genistein nor the tyrosine phosphatase inhibitor dephostatin prevented inhibition by oxotremorine-M. Hence protein kinases are not needed. Experiments with a high concentration of wortmannin were consistent with recovery being partially dependent on PIP(2) resynthesis. Wortmannin did not prevent muscarinic inhibition. Our studies of muscarinic inhibition of erg current suggest a role for phospholipase C, but not the classical downstream messengers, such as PKC or a calcium transient.
Hirdes
Wiebke
W
Department of Physiology and Biophysics, University of Washington School of Medicine, G-424 Health Sciences Building, Box 357290, Seattle, WA 98195-7290, USA.
Horowitz
Lisa F
LF
Hille
Bertil
B
D000818:Animals; D002460:Cell Line; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; D006801:Humans; D018721:Muscarinic Agonists / Q000494:pharmacology; D010095:Oxotremorine / Q000494:pharmacology; D015221:Potassium Channels / Q000502:physiology*; D024642:Potassium Channels, Voltage-Gated; D051381:Rats; D043643:Receptor, Muscarinic M1 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000502:physiology*; D043587:Receptor, Muscarinic M3 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000502:physiology*
D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; D018721:Muscarinic Agonists; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D043643:Receptor, Muscarinic M1; D043587:Receptor, Muscarinic M3; D010095:Oxotremorine