Localization and function of the Na⁺/Ca²⁺-exchanger in normal and detubulated rat cardiomyocytes

It is controversial whether the Na⁺/Ca²⁺-exchanger (NCX) can induce cardiomyocyte contraction through reverse-mode exchange and Ca²⁺-induced Ca²⁺ release (CICR). Information about the spatial distribution and functional activity within different sarcolemmal (SL) regions could shed light on this potential role. We raised a new antibody to the NCX and showed by confocal laser scanning microscopy (CLSM) that immunoreactivity is strongly expressed throughout the surface SL and intercalated disk regions with punctate labeling of the vertical transverse (T)-tubules but not the longitudinal T-tubules. Immuno-electron microscopy confirmed CLSM observations. Gold particles associated with the exchanger were within nanometer range of particles signaling ryanodine receptors. A similar close association was found between the L-type Ca²⁺ channel (known to be concentrated in the dyad) and ryanodine receptors. In whole-cell patch-clamped cardiomyocytes, peak I_NCX (measured at 90 mV) decreased by ∼40% (497 ± 32 vs. 304 ± 12 pA, P < 0.001) after detubulation, while membrane capacitance decreased by 27% (204 ± 11 vs. 150 ± 7 pF, P < 0.01) thus giving a small but significant 16% reduction in current density. Thus, the density and/or functional activity of the NCX is greater in the vertical T-tubules than in the longitudinal T-tubules, surface SL or disk regions, pointing to important functional differences between these plasma membrane domains. Our combined co-immunolocalization and physiological data suggest that the NCX has multiple functions depending upon membrane location. We suggest the possibility that NCX modulates CICR, sarcoplasmic reticulum Ca²⁺ load, and that it also serves to regulate Ca²⁺ handling in neighboring cells.