Transition states of the high-affinity rabbit Na⁺/glucose cotransporter SGLT1 as determined from measurement and analysis of voltage-dependent charge movements

Abstract

The charge-membrane voltage (Q-V) distribution of wild-type rabbit Na(+)/glucose transporter (rSGLT1) expressed in Xenopus oocytes was investigated in the absence of glucose, using the two-electrode voltage-clamp technique. Although this distribution is generally believed to be well represented by a two-state Boltzmann equation, we recently provided evidence for the existence of at least four states (Krofchick D and Silverman M. Biophys J 84: 3690-3702, 2003), confirming an earlier finding for human SGLT1 (Chen XZ, Coady MJ, and Lapointe JY. Biophys J 71: 2544-2552, 1996). We now extend our study of rSGLT1 pre-steady-state currents, employing high-resolution measurement and analysis of the Q-V distribution. A ramp, instead of a step, voltage change was used to prevent saturation of the apparatus in the first approximately 1 ms. Transient currents were integrated out to 150 ms, instead of the standard 50-100 ms. Measurements were taken every 10 mV instead of the standard 20 mV. The Q-V distribution was fit with a two-, three-, and four-state Boltzmann equation and was described best by the three-state equation. The three-state fit produced two valences of 0.45 and 1.1 at two V(0.5) values of -48 and -7.7, respectively. Our findings are critically compared with other published studies and the differences are discussed. An implication of the three-state fit is that the turnover rate of rSGLT1 is 34 s(-1), i.e., 54% greater than previously reported (22 s(-1)). Our new findings support the concept that the sugar-free model of SGLT1 is more complex than generally accepted, most likely involving a minimum of four transition states.