Topology of a functionally important region of the cardiac Na+/Ca2+ exchanger.

The cardiac Na+/Ca2+ exchanger, NCX1, has been modeled to consist of 11 transmembrane segments and a large cytoplasmic loop (loop f). Cysteine mutagenesis and sulfhydryl modification experiments demonstrate that the loop connecting transmembrane segments 1 and 2 (loop b) is located on the cytoplasmic side of the membrane, as previously modeled. A mutation in loop b, asparagine 101 to cysteine (N101C), renders the exchanger insensitive to regulation by cytoplasmic Na+ and Ca2+. Nearby mutations at residue threonine 103 (T103C or T103V) increase the apparent affinity of the exchanger for cytoplasmic Na+ and also produce a significant Li+ transport capacity. The evidence suggests that the region at the interface of cytoplasmic loop b and transmembrane segment 2 is important in Na+ transport and also in secondary regulation. Thus, this region may form part of the link between the ion translocation pathway formed by the transmembrane segments and regulatory sites that have previously been localized to loop f.

The action of Na+ as a cofactor in the inhibition by cytoplasmic protons of the cardiac Na(+)-Ca2+ exchanger in the guinea-pig.

1. Na(+)-Ca2+ exchange current was activated in giant excised patches of guinea-pig cardiac sarcolemma by raising the intracellular sodium concentration ([Na+]i). When the pHi was simultaneously acidified to 6.4, the current was transient, dropping by 80% in 30 s. 2. Pre-exposure to a pHi of 6.4 for 15 s reduced the peak Na(+)-Ca2+ exchange current without altering the decay rate or steady-state current. Recovery from proton inhibition was seen when [Na+]i was removed for 9 s. 3. A mathematical model of Na(+)-Ca2+ exchange function reproduced the experimental results. In addition, two model-dependent predictions were seen experimentally. (i) [Na+]i-dependent 'inactivation' of Na(+)-Ca2+ exchange may arise from pHi effects. We observed experimentally that pre-exposure to acidic pHi can remove the transient current component attributed to [Na+]i-dependent 'inactivation'. (ii) self-exchange should be inhibited by acidification. This has been observed by other investigators. 4. We have hypothesized that there are two components to inhibition of the Na(+)-Ca2+ exchanger by intracellular protons, and that one is enhanced by increased [Na+]i (Doering & Lederer, 1993b). This hypothesis is supported by the data presented here and by a model of Na(+)-Ca2+ exchange behaviour in which binding of intracellular sodium to the exchanger enhances the affinity of the exchanger for inhibitory intracellular protons.

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)