Alkaline pH facilitates the exchange of guanine nucleotides: A possible mechanism for modulation of the kinetics of responses mediated by guanine nucleotide-binding proteins

Abstract

GTP gamma S1 binding experiments in a particulate preparation from Xenopus oocytes revealed two binding sites at pH = 6.9: A high affinity site (Kd = 77 +/- 4 nM) and a low affinity site (Kd = 8.74 +/- 0.05 microM). Alkaline pH (8.5) caused a significant increase in the dissociation constants of both sites (160 +/- 46 nM and 30.7 +/- 1.6 microM, respectively). In purified plasma membrane preparation, alkaline pH increased the rate of dissociation of GTP gamma S. We have previously proposed that the activation of a G-protein by the agonist-occupied receptor is rate-limiting in the kinetics of hormone-induced responses (Lipinsky et al., 1993; Pflugers Arch., 425:140-149). We have, therefore, assayed the latencies of responses evoked by TRH at different pH, in oocytes expressing the TRH receptor. A change in the medium pH was reflected by an approximately tenfold smaller change in cellular pH (pHi). Alkalinization of the medium (from pH 7.4 to 8.5) caused a shortening of latency (by 45%), whereas acidification to pH = 6.0 prolonged it (by 87%). Moreover, alkalinization decreased the latency and increased the rate of responses to microinjected GTP gamma S, but did not change the latency of responses to microinjected InsP3. These results show that activation of plasma membrane receptors coupled to G-proteins, concurrent with a change in pHi, can alter the kinetic pattern of physiological responses, thus affecting the ultimate physiological output of the cell. This finding suggests that a change of pH, is a novel potential mechanism for modulation of responses mediated by G-proteins.

Menthol Inhibits 5-HT3Receptor–Mediated Currents

“…[ (Lipinsky and Oron, 1996). One hundred fifty oocytes were gently homogenized at 4°C by passing through a 27-gauge needle in 4 ml of homogenization medium [5 mM NaCl, 5 mM Na-HEPES, 0.6 mM PMSF, 4 pM leupeptin (pH 7.5)].…”