FAST VOLTAGE-CLAMP DISCLOSES A NEW COMPONENT OF PRESTEADY STATE CURRENT FROM THE NA-GLUCOSE COTRANSPORTER()

The human Na+-glucose cotransporter (hSGLT1) has been shown to generat e, in the absence of sugar, presteady-state currents in response to a change in potential, which could be fitted with single exponentials on ce the voltage had reached a new constant value. By the cut-open oocyt e technique (voltage rising-speed similar to 1 mV/mu s), phlorizin-sen sitive transient currents could be detected with a higher time resolut ion during continuous intracellular perfusion. In the absence of sugar and internal Na+, and with 90 mM external Na+ concentration ([Na+](o) ), phlorizin-sensitive currents exhibited two relaxation time-constant s: tau(1) increased from 2 to 10 ms when V-m decreased from +60 mV to -80 mV and remained at 10 ms for more negative V-m; tau(2) ranged from 0.4 to 0.8 ms in a weakly voltage-dependent manner. According to a pr eviously proposed model, these two time constants could be accounted f or by 1) Na+ crossing a fraction of the membrane electrical field to r each its binding site on the carrier and 2) conformational change of t he free carrier, To test this hypothesis, the time constants were meas ured as [Na+](o) was progressively reduced to 0 mM. At 30 and 10 mM ex ternal Na+, tau(1) reached the same plateau value of 10 ms but at more negative potentials (-120 and -160 mV, respectively). Contrary to the prediction of the model, two time constants continued to be detected in the bilateral absence of Na+ (at pH 8.0), Under these conditions, t au(1) continuously increased through the whole voltage range and did n ot reach the 10 ms level even when V-m had attained -200 mV while tau( 2) remained,in the range of 0.4-0.8 ms. These results indicate that 1) conformational change of the free carrier across the membrane must oc cur in more than onestep and 2) Na+ binding/debinding is not responsib le for either of the two observed exponential components of transient currents. By use of the simplest kinetic model accounting for the port ion of the hSGLT1 transport cycle involving extracellular Na+ binding/ debinding and the dual-step conformational change of the free carrier, tau(1) and tau(2) were fitted throughout the voltage range, and a few sets of parameters were found to reproduce the data satisfactorily. T his study shows 1) tau(1) and tau(2) correspond to two steps in the co nformational change of the free carrier, 2) Na+ binding/debinding modu lates the slow time constant (tau(1)), and 3) a voltage-independent sl ow conformational change of the free carrier accounts for the observed plateau value of 10 ms.