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ABSTRACT
Formamide-induced detubulation of rat ventricular myocytes was used to investigate the functional distribution of the Na/Ca exchanger (NCX) and Na/K-ATPase between the t-tubules and external sarcolemma. Detubulation resulted in a 32% decrease in cell capacitance, whereas cell volume was unchanged. Thus, the surface-to-volume ratio was used to assess the success of detubulation. NCX current (I^sub NCX^) and Na/K pump current (I^sub pump^) were recorded using whole-cell patch clamp, as Cd-sensitive and K-activated currents, respectively. Both inward and outward I^sub NCX^ density was significantly reduced by ~40% in detubulated cells. I^sub NCX^ density at 0 mV decreased from 0.19 ± 0.03 to 0.10 ± 0.03 pA/pF upon detubulation. I^sub pump^ density was also lower in detubulated myocytes over the range of voltages (-50 to +100 mV) and internal [Na] ([Na]^sub i^) investigated (7-22 mM). At [Na]^sub i^ = 10 mM and -20 mV, I^sub pump^ density was reduced by 39% in detubulated myocytes (0.28 ± 0.02 vs. 0.17 ± 0.03 pA/pF), but the apparent K^sub m^ for [Na]^sub i^ was unchanged (16.9 ± 0.4 vs. 17.0 ± 0.3 mM). These results indicate that although the t-tubules represent only ~32% of the total sarcolemma, they contribute ~60% to the total I^sub NCX^ and I^sub pump^. Thus, the functional density of NCX and Na/K pump in the t-tubules is 3-3.5-fold higher than in the external sarcolemma.
INTRODUCTION
The sarcolemma of mammalian cardiac ventricular myocytes has invaginations (transverse (t-) tubules) that occur periodically at the level of the Z-line, and comprise 21-64% of the total sarcolemmal membrane area (Page et al., 1971; Page and Surdyk-Droske, 1979; Soeller and Cannell, 1999; Bers, 2001). Immunocytochemical studies have shown that L-type calcium channels are concentrated in the t-tubules, in close apposition to the ryanodine receptor (Carl et al., 1995; Scriven et al., 2000) at the dyadic junction between the sarcolemma and the sarcoplasmic reticulum (SR) membrane. This allows the fast inward spread of excitation-contraction coupling so that SR Ca release is spatially and temporally synchronized throughout the cell (Yang et al., 2002; Brette et al., 2002; Bers, 2001). In contrast, in atrial and neonatal ventricular myocytes, and Purkinje cells, which lack appreciable t-tubules, [Ca]^sub i^ rises first at the edge of the cell and then propagates into the cell interior...