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

1. 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)