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1 Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada
2 Department of Medicine, University of Toronto, Toronto, Ontatio, Canada
* To whom correspondence should be addressed. E-mail: melvin.silverman{at}utoronto.ca.
The charge, Q, versus membrane voltage, V, distribution of wt 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, recently, we provided evidence for existence of at least four states, confirming an earlier finding for hSGLT1. We now extend our study of rSGLT1 pre-steady-state currents, employing high resolution measurement and analysis, of the Q versus V distribution. A ramp, instead of a step, voltage change was used to avoid 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 instead of the standard 20 mV. The Q versus 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 V0.5's 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, likely involving a minimum of four transition states.
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