Fig. 4. Electrophysiological analysis of HMIT transport activity. (A) Original current recording from a Xenopus oocyte expressing HMIT, at a holding potential of –50 mV, and in a pH 5.0 extracellular solution. Addition of 2 mM myo-inositol (In.) induced a reversible inward current in a Na⁺-containing or a nominally Na⁺-free solution (no Na). I–V curves were recorded before, during and after each exposure to myo-inositol. (B) I–V curves of the current induced by 2 mM myo-inositol, at pH 5.0, in non-injected oocytes (open symbols, n = 5) and in oocytes expressing HMIT (closed symbols, n = 18). Currents were recorded in Na⁺-containing solution (circles) and in nominally Na⁺-free solutions (triangles). In non-injected oocytes, myo-inositol had no significant effect on the holding current at any membrane potential. In oocytes expressing HMIT, an inward current was activated by myo-inositol; the amplitude of this current was slightly (∼15%) smaller in the nominal absence of Na⁺ that in the presence of 100 mM Na⁺. (C) I–V curves of the current induced by 2 mM myo-inositol, at pH ranging from 4.0 to 7.5 in oocytes expressing HMIT (n = 7). At all membrane potentials, the amplitude of the current induced by 2 mM myo-inositol increased from practically 0 at pH 7.5 to a maximum at pH 4.0. At high negative membrane potential, there was no increase between pH 5.0 and 4.0, indicating saturation. (D) Quantification of the pH dependence of inositol transport. The best fitting I_max and pK_a were obtained by fitting the following equation to the inositol-induced current (I_in) at each potential, using the non-linear fit routine of the Kaleidagraph® program (Synergy Software, Reading, PA): I_in = I_max/10^{(pH – pKa)}. Examples of fits are presented for the –110, –50 and 10 mV membrane potentials. (E) The pK_a and the I_max calculated for each membrane potential, from the mean current values at the five pH values tested. Both the maximal current and the apparent pK_a were voltage sensitive (large maximal currents and lower pK_a at negative potentials), with a steeper slope for pK_a.