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Free keywords:
DM-nitrophen; caged Ca2+; antporter
Abstract:
The Na/Ca exchanger catalyzes the countertransport of Na+ and Ca2+ as well as the self-exchange of either Na+ or Ca2+, that is, Na/Na and Ca/Ca exchange. These self-exchange modes offer a unique possibility to investigate partial reactions of the exchanger. Previously, using a combination of patch-clamp techniques and photolytic Ca2+ concentration jumps, we showed that the exchanger generates a transient current under conditions promoting Ca/Ca exchange when the Ca2+ concentration on the cytolosolic side is rapidly increased. Thus, contrary to previous suggestions, charge translocation is involved in Ca2+ translocation, although stationary Ca/Ca exchange is electroneutral. A cytosolic Ca2+ jump generates inward, not outward, current. Thus, it has been suggested that the movement of overall negative charge is correlated with the outward translocation of Ca2+. The time course of the current signal is fairly rapid: time to peak is less than 0.1 ms, and the duration is <1 ms. Thus, very fast Ca2+ concentration jumps and recording techniques are required if this signal is to be analyzed quantitatively. Photoinduced Ca2+ release from the chelator DM-nitrophen occurs with at least 38,000 s−1 (2.6 μs) and the bandwidth of the patch clamp is about 10 kHz (15 ms). To learn more about the mechanism of Ca2+ translocation, we investigated the voltage and Ca2+ dependence of the transient current. Initial experiments were performed with membrane patches excised from guinea pig cardiac myocytes. More detailed investigations were conducted with NCX1 from guinea pig expressed in Xenopus oocytes. No significant differences between either system have been observed so far. A major advantage of the oocyte system is that on the average, signal amplitudes are much larger than ones obtained with membranes from cardiac myocytes.
