The mechanism by which cytoplasmic protons inhibit the sodium‐calcium exchanger in guinea‐pig heart cells.

We recorded cardiac sodium-calcium exchange current (INa-Ca) in giant excised membrane patches obtained from cardiac myocytes of the adult guinea-pig. 2. Rapid changes in ion concentrations on the cytoplasmic side of the excised membrane patch were produced using a modified oil-gate bath. 3. Sodium-calcium exchange current was activated by step increases in sodium concentration on the cytoplasmic side of the membrane (Na+i), which led to an increase in outward INa-Ca to a new steady-state level. The Na+i required to half-maximally activate the sodium-calcium exchange current (K1/2) was 21 mM. 4. Step increases in cytoplasmic calcium concentration (Ca2+i) stimulated the Na+i-activated INa-Ca up to 1 microM Ca2+i, then inhibited the exchange current at very high Ca2+i (1 mM). 5. A step decrease in cytoplasmic pH from 7.2 to 6.4 (increase in H+i) produced a biphasic but monotonic decrease in INa-Ca. Alkalinization of cytoplasmic pH from 7.2 to 8.0 caused a large, biphasic increase in INa-Ca. 6. When INa-Ca was activated by a step increase in Na+i and H+i was simultaneously increased, the outward current rose to a peak and then declined to a low steady level. The peak current seen was always less than the maximum current produced by an identical elevation of Na+i at constant pHi. This reduction in peak outward current reflected a rapid 'primary' inhibition of the sodium-calcium exchange by protons. The decay of the sodium-calcium exchange current following the peak was slow and corresponded to the time course of the onset of a 'secondary' proton block. 7. Rapid primary inhibition of the sodium-calcium exchanger could also be produced by cytoplasmic acidification in the absence of cytoplasmic sodium. The primary blockade was revealed when a subsequent increase in Na+i activated INa-Ca and a smaller peak outward current was observed. Secondary inhibition of the sodium-calcium exchanger was not, however, produced by cytoplasmic acidification in the absence of cytoplasmic sodium. Regardless of the duration of exposure to elevated H+i, the 'secondary' block by protons was still seen on activation of INa-Ca by increased Na+i as a gradual reduction of outward current amplitude. 8. Treatment of the sodium-calcium exchanger with the proteolytic enzyme alpha-chymotrypsin largely removed its sensitivity to protons. 9. We conclude that the action of alpha-chymotrypsin on the monomeric sodium-calcium exchange protein is in part to remove a proton-sensitive regulatory component(s) or render the regulation ineffective.(ABSTRACT TRUNCATED AT 400 WORDS)