Pipette GTP is essential for receptor-mediated regulation of Cl- current in dialysed myocytes from guinea-pig ventricle.
1. Wide-tipped, low-resistance (approximately 1 M omega) pipettes were used to record the whole-cell Cl- current activated by cAMP-dependent protein kinase (PKA) in guinea-pig ventricular myocytes internally dialysed with or without GTP. Without GTP in the pipette, the response to 1 microM-isoprenaline declined with time and eventually disappeared, usually within approximately 20 min of rupturing the membrane and beginning cell dialysis. 2. This rundown of the isoprenaline response occurred more quickly with wider, lower-resistance pipette tips. 3. After complete rundown of the isoprenaline response, histamine (10 microM), another agonist known to elicit the Cl- current, also had no effect, but extracellular forskolin (1 microM) or intrapipette cAMP (1 mM) could still readily elicit the Cl- current. 4. In contrast, with 100 microM-GTP in the pipette, the response to 1 microM-isoprenaline was well maintained for periods greater than 20 min. But, if GTP was then withdrawn from the pipette, a rundown of the isoprenaline response was seen comparable to that in the experiments begun with GTP-free pipette solution. Moreover, in experiments begun without pipette GTP, the addition of 100 microM-GTP to the pipette solution, after the response to isoprenaline had disappeared, was able to restore that Cl- current response. 5. With GTP in the pipette, the forskolin-induced Cl- current could be suppressed by concurrent exposure to carbachol (10 microM). That inhibition was not seen in myocytes pretreated with pertussis toxin. In untreated myocytes dialysed with GTP-free pipette solution, after disappearance of the isoprenaline response, the muscarinic receptor-mediated inhibition was itself abolished. 6. We confirm that both beta-adrenoceptor-mediated activation of the Cl- current by isoprenaline, and muscarinic receptor-mediated inhibition of the forskolin-induced Cl- current, are mediated by G proteins, and conclude that the disappearance of both receptor-mediated responses during whole-cell recording with GTP-free pipette solution reflects the fall of cellular [GTP] below the level required to maintain G protein-dependent signal transduction.
Horie
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Laboratory of Cardiac/Membrane Physiology, Rockefeller University, New York, NY 10021.
Hwang
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Gadsby
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D000818:Animals; D002463:Cell Membrane Permeability / Q000502:physiology; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006168:Guinea Pigs; D008565:Membrane Proteins / Q000502:physiology; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism; D016276:Ventricular Function
D018118:Chloride Channels; D002712:Chlorides; D008565:Membrane Proteins; D006160:Guanosine Triphosphate