The membrane potential (ΔΨ) and the pH gradient (ΔpH) across the membrane of the insulin-secretory granule were determined in studies in vitro from the uptake of the permeant anion thio[14C]cyanate or the permeant base [14C]methylamine. Freshly prepared granules incubated in iso-osmotic medium containing sucrose and low concentrations of buffer salts exhibited an acidic internal pH and a ΔΨ positive inside. Addition of MgATP2− under these conditions did not alter the ΔpH, but produced a marked increase in the ΔΨ. Conversely, when a permeant anion was also included, ATP produced a marked increase in the ΔpH and a lesser increment in the ΔΨ. NH4+ salts reduced the ΔpH across granule membranes. In the presence of ATP this effect was accompanied by a reciprocal increase in the ΔΨ. A similar reciprocity was evident when nigericin was added together with K+ or on decreasing the medium pH, suggesting that these gradients were linked by a common electrogenic process. The effects of ATP were reversed by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the combination of valinomycin, nigericin and K+, and by the Mg2+-dependent ATPase inhibitor tributyltin. Uptakes of 14C-labelled tracer molecules were also markedly reduced by cryogenic disruption of the granule membrane or hypo-osmotic incubation conditions. These results were readily interpreted within a chemiosmotic hypothesis, which proposed that the insulin granules possess an inwardly-directed electrogenic proton-translocating Mg2+-dependent ATPase with the additional postulate that the membrane has a low proton permeability. The intragranular pH was estimated as being between 5 and 6 in vivo. Such a value corresponds to optimal conditions for the crystallization of zinc–insulin hexamers. Several other functions related to chemiosmotic processes within insulin granules, however, may be envisaged.
Research Article| April 15 1982
The internal pH and membrane potential of the insulin-secretory granule
Biochem J (1982) 204 (1): 171–178.
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John C. Hutton; The internal pH and membrane potential of the insulin-secretory granule. Biochem J 15 April 1982; 204 (1): 171–178. doi: https://doi.org/10.1042/bj2040171
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