Temperature dependence of cloned mammalian and salmonid cardiac Na+/Ca2+ exchanger isoforms

The cardiac Na+/Ca2+ exchanger (NCX), an important regulator of cytosolic C a2+ concentration in contraction and relaxation, has been shown in trout he art sarcolemmal vesicles to have high activity at 7 degreesC relative to it s mammalian isoform. This unique property is likely due to differences in p rotein structure. In this study, outward NCX currents (I-NCX) of the wild-t ype trout (NCX-TR1.0) and canine (NCX 1.1) exchangers expressed in oocytes were measured to explore the potential contributions of regulatory vs. tran sport mechanisms to this observation. cRNA was transcribed in vitro from bo th wild-type cDNA and was injected into Xenopus oocytes. I-NCX of NCX-TR1.0 and NCX1.1 were measured after 3-4 days over a temperature range of 7-30 d egreesC using the giant excised patch technique. The I-NCX for both isoform s exhibited Na+-dependent inactivation and Ca2+-dependent positive regulati on. The I-NCX of NCX1.1 exhibited typical mammalian temperature sensitiviti es with Q(10) values of 2.4 and 2.6 for peak and steady-state currents, res pectively. However, the I-NCX of NCX-TR1.0 was relatively temperature insen sitive with Q10 values of 1.2 and 1.1 for peak and steady-state currents, r espectively. I-NCX current decay was fit with a single exponential, and the resultant rate constant of inactivation (lambda) was determined as a funct ion of temperature. As expected, lambda decreased monotonically with temper ature for both isoforms. Although lambda was significantly greater in NCX1. 1 compared with NCX-TR1.0 at all temperatures, the effect of temperature on lambda was not different between the two isoforms. These data suggest that the disparities in I-NCX temperature dependence between these two exchange r isoforms are unlikely due to differences in their inactivation kinetics. In addition, similar differences in temperature dependence were observed in both isoforms after a-chymotrypsin treatment that renders the exchanger in a deregulated state. These data suggest that the differences in I-NCX temp erature dependence between the two isoforms are not due to potential dispar ities in either the I-NCX regulatory mechanisms or structural differences i n the cytoplasmic loop but are likely predicated on differences within the transmembrane segments.