Permeant anion activation of MgATPase activity in chromaffin granules. Evidence for direct coupling of proton and anion transport.

Abstract

Osmotic lysis of chromaffin granules, due to the combined activities of a proton-pumping MgATPase and an anion transport site in its membrane, is blocked by anion transport inhibitors such as 4-acetamido-4.4~0thiocyanostilbene-2,2'-disulfonicacid (SITS) and pyridoxal phosphate by competition with chloride ions (Pazoles, C. J., and Pollard, H. B. (1978) J. Biol.Chem 253, 3962-3969).We have now discovered that these compounds also inhibit the granule ATPase activity, and we therefore questioned whether the inhibitors acted primarily at the anion transport site or at the proton pumping granule ATPase, or whether the ATPase itself was perhaps somehow directly coupled to anion-sensitive sites.We found that the ATPase activity could indeed be stimulated by permeant anions (Cl-, Br-) but not impermeant ones (isethionate, phosphate).The differential effects of anions and inhibitors on this activity were observed with intact, water-lysed, and detergentsolubilized granules.The influence of these substances on granule lysis was not, however, necessarily linked to ATP hydrolysis since their effects could still be observed when MgATP was omitted and an artificial inward proton gradient put in its place.Other ATP-independent mechanisms of granule lysis showed that anion transport inhibitors were effective at blocking lysis only when the mechanism involved co-transport of both anions and protons.Measurements of the granule transmembrane potential further demonstrated that the inhibitors were capable of selectively inhibiting chloride as opposed to proton transport.We have interpreted these data to indicate that the protonpumping MgATPase and the anion transport site may exist as parts of a macromolecular complex within the granule membrane.Chromaffin granules isolated from the bovine adrenal medulla undergo osmotic lysis when exposed to MgATP and perrneant anions, such as chloride.Lysis occurs as a consequence of ATPase-driven proton uptake and the concomitant uptake of perrneant anions via a transport site having pharmacological similarities to the anion transport site of erythrocytes (1, 2).These similarities are based on observations that drugs such as SITS,' pyridoxal phosphate, probenecid, and others inhibit granule lysis by competing with chloride in