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

1 Institute of Medical Science and 2 Department of Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8 Submitted 6 January 2004 ; accepted in final form 11 February 2004 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 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. SGLT1; Boltzmann distribution; Xenopus oocyte; sodium/glucose cotransport; two-electrode voltage clamp Address for reprint requests and other correspondence: M. Silverman, Dept. of Medicine, Univ. of Toronto, Toronto, Ontario, Canada M5S 1A8 (E-mail: melvin.silverman{at}utoronto.ca ).