Abstract
The sided effects of H+ 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 (DIDS), which inhibits pH equilibration across the membrane. This treatment provides a "pH clamp" whereby the internal and external H+ (H+i and H+o) concentrations can be varied separately. Channel activity was evaluated by measuring either net K+ loss or unidirectional 42K+ efflux from cells where SO2(-4) replaced Cl- on both sides of the membrane. When pHi was set at 7.4, decreasing pHo from values of 8.0 to 5.0 inhibited K+ efflux. This effect of H+o could be overcome by increasing K+o at all values of pHo. In addition, this effect of K+o could be separated from its effects on altering the membrane potential, indicating an interaction between K+o and H+o on the channel. A similar interaction was shown to occur between H+i and K+i. K+o is known to be required for activation of Ca2(+)-stimulated K+ transport, since the channel in cells preincubated in the absence of K+o (prior to exposure to Ca+i) becomes refractory to subsequent activation by Ca2+i and K+o. We found that H+o would not substitute for K+o in this regard nor would H+o inhibit the protective effect of K+o; in addition, H+ was not transported inward in exchange for K+i. Thus it would appear that there are two external sites where K+o interacts with the channel. One site is antagonized by H+o, whereas the second site is required for channel activation independent of H+ in the range studied. The inside of the channel would have, by an analogous argument, at least one site where K+i and H+i interact.