Abstract
Aluminum fluoride (AlF4-) activates the heterotrimeric G protein Gs (stimulatory G protein of adenylylcyclase) (Sternweis, P. C., and Gilman, A. G. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 4888-4891) and GT (transducin), and for GT, Bigay et al. (Bigay, J., Deterre, P., Pfister, C., and Chabre, M. (1985) FEBS Lett. 191, 181-185) have made the intriguing proposal that AlF4- acts by mimicking the gamma-phosphate of GTP. The endogenous G protein (probably G alpha i-2 or G alpha i-3 (Yatani, A., Mattera, R., Codina, J., Graf, R., Okabe, K., Padrell, E., Iyengar, R., Brown, A. M., and Birnbaumer, L. (1988) Nature 336, 680-682) that stimulates the muscarinic atrial K+ (K+[ACh]) channel is also thought to be activated by AlF4- (Kurachi, Y., Nakajima, T., and Ito, H. (1987) Circulation 76, 105P). To investigate the AlF4- mechanism, we applied potassium fluoride (KF) to the cytoplasmic face of inside-out membrane patches excised from guinea pig atria. We found that KF activated single K+[ACh] channel currents in both a concentration- and a Mg(2+)-dependent manner. Activation persisted following removal of KF, but unlike activation by guanosine 5'-(3-thiotriphosphate) (GTP gamma S), was fully reversed by removal of Mg2+. Evidence for Al3+ involvement was that the Al3+ chelator deferoxamine (500 microM) inhibited KF activation and that at low concentrations of KF (less than 1 mM), micromolar AlCl3 concentrations potentiated KF stimulation. The rate of activation produced by KF was far slower than the rate produced by GTP or GTP gamma S, and unlike these guanine nucleotides, the rate was unchanged in the presence of agonist. To test the gamma-phosphate-mimicking hypothesis, we evaluated the requirement for GDP; and to accomplish this, it was necessary to establish a condition that ensured exchange of guanine nucleotides. This condition was satisfied by using the muscarinic agonist carbachol because both the rate and the extent of activation of the K+[ACh] channels produced by GTP were much faster in carbachol, and both were greatly slowed when GDP was added along with GTP. By contrast, the effects of KF were unchanged by carbachol in the presence or absence of GDP. Further evidence that GDP is not essential for activation by AlF4- was provided by the observation that during carbachol activation and following extensive washing with GMP, guanosine 5'-O-(2-thiodiphosphate) at blocking concentrations had no effect on activation produced by KF.(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
Lett. 191,181-185) have made the intriguing proposianl guinea pig atrium [1].The responsible G protein is called that Aluminum fluoride (AlF); acts by mimicking the y-phosphate of GTP
NaF activates transducin and the G proteins responsible for polyphosphoinositide hydrolysis [12,13,14]; and pertinent to our interest in ion channels as G protein effectors, an abstract has been published indicating that AlF; may activate Gk[15]
Activation by AlF; is especially intriguing because it has been proposed that AlF; activates the a subunit of G proteins
Summary
Single atrial cells were dissociated from adult guinea pig heart by collagenase digestion [3]. MgCI, and KF were added to the bathing solution; open times in KF- and GTP-activated channels at -80mV when KF concentrations >10 mM were tested, KC1 was replaced by were1.3 k 0.3 and 1.2 k 0.5 ms, respectively. KF activated K+[ACh]channel currents to thseame extent in the presence or absence of the muscarinic agonist carbachol (10 p ~ in) the patch solution (Fig. 4B). Current-voltage relationships of KF-and GTP-activated A1C13 at 100 p~ enhanced the activation by submaximum channels were indistinguishable (Fig. IC, panel 3 ) , and the concentrations of KF at 1 mM by 40 k 8% ( n = 5) in the single channel slope conductances of inward channel current presence of 2 mM Mg2+.A t 20 mM M$+, A1C13 (100 p ~ha)d were 38 -+ 1picosiemens ( n = 4 for each condition). No significant of single channel currentsin eight patches showed that mean potentiation by A1C13 was obtained at concentrations of KF
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