Membrane sidedness and the interaction of H+ and K+ on Ca2(+)-activated K+ transport in human red blood cells.

Abstract

The sided effects of HI on Ca2+-stimulated K+ transport (the Gardos channel) were studied in human red blood cells. Cells were loaded with Ca2' during energy depletion with the internal pH adjusted to desired levels prior to treatment with the anion-exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DWDS), which inhibits pH equilibration across the membrane. This treatment provides a "pH clamp" whereby the internal and external HI (Hit and Since Ca2l-activated K+ channels in human red blood cells were first described by Gardos (1), much has been learned about the properties of these channels, including the dependencies on intracellular Ca2l (Ca; +) and on K+ on both sides of the membrane (Kt and K') (2-4). Analysis of the channel's conductance by use of the patch-clamp technique (5-8) and indirect methods (9, 10) has helped to define various channel characteristics, including that the channel operates as an inward rectifier and that K+ diffuses through it by a single-file mechanism.HoPrevious work (11, 12) on the effects of H' on the Gardos channel has been limited because, until the emergence of anion-exchange inhibitors such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) (13), it was not possible to separately control inside and outside variations in pH. Nevertheless, Porzig (11) MATERIALS AND METHODSPreparation of Cells. Fresh heparinized human blood was obtained from healthy donors. To study Ca2l-stimulated K+ transport, depletion of the cells' ATP is necessary so that whatever Ca2+ taken up will be retained for a period of time sufficient to measure K+ efflux. Thus, the red cells were washed three times by centrifugation (15,000 X g) at 40C, with 10 volumes of the incubation medium that was also used for concomitant loading with Ca2+ and depletion of their endogenous energy sources. Ca2+ loading was carried out in two ways. The first way used the salicylate method (20) where the cells were also energy-depleted (21) by incubation for 2.5 hr at 370C in a medium that contained 5 mM inosine and 5 mM iodoacetamide in addition to 5mM CaCl2, 60 mM KCl, 15 mM Pipes, and 75 mM salicylic acid, brought to pH 7.5 with KOH.The second way was by incubation of the cells at 370C in the presence of either CaSO4 or CaCl2 (up to 5 mM) in SO2-or Cl-solutions that contained either 50 mM K2SO4 plus 40 mM Na2SO4 or 140 mM KCI plus 10 mM NaCl, each being buffered with 30 mM Mops or Hepes at pH 7.5 and containing 5 mM inosine plus 5 mM iodoacetamide for energy depletion.The cells were suspended at 40%o hematocrit and incubated at 370C for 2.5-4 hr. Afterwards the cells in each case were washed three or four times with 20 volumes of the medium (40C) into which the efflux was to be measured. The composition of the relevant flux media is specified in the legends.In some experiments Ca2`-induced K+ transport was studied in reconstituted ghosts (22) 1998The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement"...

Phosphate from the phosphointermediate (EP) of the human red blood cell Na/K pump is coeffluxed with Na, in the absence of external K.

“…7). The results presented in Table V show that, in intact cells, when the pHi is clamped at ~7.3 (by the use of DIDS treatment [see Heinz and Hoffman, 1990]), lowering the pHo to 6.2 inhibits the effluxes of both Pi and SO4. (In the two instances shown, the SO4 efflux is not significantly different from zero.)…”

“…The pHo and pHi values shown were determined on aliquots removed after the cells had been added to their respective media just before and after the 35 rain flux period was started. The pH's given in parentheses represent the latter values and indicate the effectiveness of the pH clamp (see Heinz and Hoffman, 1990). The ettluxes were measured in the absence and presence of 100 IsM ouabain but only the ouabain-sensitive components are presented.…”

“…The pHo and pHi values shown were determined on aliquots removed after the cells had been added to their respective media just before and after the 35 rain flux period was started. The pH's given in parentheses represent the latter values and indicate the effectiveness of the pH clamp (see Heinz and Hoffman, 1990) Kennedy et al (1986) found that the onset of inhibition of onabain-sensitive Nai/Ko exchange was already apparent when the ratio of the levels of ADP/ATP were equal to or greater than one. This also applies to the uncoupled ol~A ouab olMro uab and efflux mode, as seen in Table VI, when partial inhibition of ""Ya ....…”