Na/Ca exchange and Na/K-ATPase function are equally concentrated in transverse tubules of rat ventricular myocytes

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(NCX)) and Na/K pump current (I(pump)) were recorded using whole-cell patch clamp, as Cd-sensitive and K-activated currents, respectively. Both inward and outward I(NCX) density was significantly reduced by approximately 40% in detubulated cells. I(NCX) density at 0 mV decreased from 0.19 +/- 0.03 to 0.10 +/- 0.03 pA/pF upon detubulation. I(pump) density was also lower in detubulated myocytes over the range of voltages (-50 to +100 mV) and internal [Na] ([Na](i)) investigated (7-22 mM). At [Na](i) = 10 mM and -20 mV, I(pump) density was reduced by 39% in detubulated myocytes (0.28 +/- 0.02 vs. 0.17 +/- 0.03 pA/pF), but the apparent K(m) for [Na](i) was unchanged (16.9 +/- 0.4 vs. 17.0 +/- 0.3 mM). These results indicate that although thet-tubules represent only approximately 32% of the total sarcolemma, they contribute approximately 60% to the total I(NCX) and I(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.

FIGURE 1

Effect of detubulation on cell surface, volume, and calcium current. (A) Cell capacitance vs. volume for all control (▪) and detubulated (○) cells studied. Mean volume and capacitance values are shown by the bigger symbol. A linear regression was calculated for control (solid line) and detubulated (dotted line) cells. Note the presence of two outlier “detubulated” cells (▵) near the control regression line. These cells were not considered for currents analysis. (B) Mean data for surface-to-volume ratio (S:V) in control (n = 18, black bar) and detubulated cells (n = 19, open bar). (C) Mean I_Ca in control (n = 22, black bar) and detubulated cells (n = 21, open bar). The right axis represents value normalized to control data. All bars are means ± SE; asterisk indicates P < 0.05.

FIGURE 2

I_NCX is concentrated in the t-tubules. (A) Representative example of the membrane current recorded in a control cell during the voltage ramp protocol (top panel) in the absence (Ctl) and in the presence (Cd) of 1 mM Cd. Free [Ca]_i in the pipette was 100 nM. (B) Current-voltage relationship for the currents shown in A during the descending ramp and the difference current (Diff). (C) Mean current density-voltage relationship of I_NCX, i.e., the Cd-sensitive current, recorded in 12 control (Ctl) and 11 detubulated (Det) myocytes. (D) Voltage dependence of the ratio between I_NCX in detubulated and control cells. (Inset) Mean I_NCX density at 0 mV in control and detubulated cells.

FIGURE 3

NCX in the sealed-off t-tubules membrane might still be functionally active. (A) Normalized traces of caffeine-induced Ca transient in a control (Ctl), detubulated (Det), and detubulated with 1 mM ouabain entrapped in the sealed-off t-tubules (Det+ouab) myocytes. All myocytes were stimulated at steady state (at 0.5 Hz) before the application of 10 mM caffeine. (B) Mean data for the decay time of caffeine-induced Ca transient in 18 control cells, 14 detubulated myocytes, and 11 detubulated cells with 1 mM ouabain.

FIGURE 4

I_pump is concentrated in the t-tubules. (A) Representative example of I_pump measured at −20 mV as the outward shift induced by 4 mM K in a control cell. (B) Mean data for I_pump in 16 control and 14 detubulated myocytes. (C) Mean I_pump-voltage relationship recorded in 8 control and 8 detubulated myocytes. (D) Voltage dependence of the ratio between I_pump in detubulated and control cells.

FIGURE 5

[Na]_i-dependence of I_pump in control and detubulated myocytes. Changes in I_pump (A and C) and [Na]_i (B and D) upon abrupt Na/K pump re-activation after 10–12 min of pump blockade in the absence of external K, in a control (A and B) and a detubulated (C and D) myocyte. (E) Mean I_pump and d[Na]_i/dt at various [Na]_i in seven control and eight detubulated myocytes. Data are fit with a Hill equation (V_max/(1+(K_m/[Na]_i)³).

FIGURE 6

The relative distribution of Ca channels, NCX, and Na/K pumps between t-tubules and external sarcolemma. (A) Percentage of the whole-cell I_Ca, I_NCX, and I_pump originating in the t-tubules and the ratio between the current density in the t-tubules and external sarcolemma. The currents in t-tubules were calculated as the difference in the whole-cell current between control and detubulated myocytes. These currents were divided by the difference in the membrane capacitance in control versus detubulated cells to derive the current density in t-tubules. (B) The mean ratio between I_Ca, I_NCX, and I_pump in control and detubulated cells. The values at 0 mV and 100 nM [Ca]_i were used for I_NCX.