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Figure 4. R255N alters the extent of S4 movement. (A and B) Currents from an excised inside-out patch containing I262C channels in response to voltage steps from −40 to 80 mV, before (A) and after (B) application of internal MTSET. 1 mM MTSET was applied for 10 s on closed channels held at −100 mV in between the two recordings. (C and F) Currents from an excised inside-out patch containing R255N/I262C channels in response to voltage steps from −40 to 100 mV (in 20-mV increments), before (C) and after (F) 1 mM MTSET was applied 10 times at −100 mV for 1 s. pHi = 5.5 and pHo = 7. (D) Currents in response to a 100-mV voltage step every 10 s during MTSET application on open R2355N/I262C channels. The speed of the perfusion of the cytosolic face of the patch was tested by applying 1 mM MTSET (in pH 7.0 solution) at 100 mV for 1 s, followed by washout at 100 mV (pH 5.5 solution). Wash-in and washout were monitored by the fast changes in current amplitude induced by changes in internal pH: pHi = 5.5 in rinse and pHi = 7.0 in MTSET solution. pHo = 7.0 (pipette solution). (E) Currents in response to a 100-mV voltage step every 10 s during MTSET application on closed R255N/I262C channels. Closed-state modification was tested by applying 1 mM MTSET at −60 mV for 1 s, followed by washout. Same patch as D. (G) Normalized currents in response to voltage steps to 100 mV after sequential 1-s applications of 1 mM MTSET applied at −100 mV on I262C (red boxes) and R255N/I262C (black boxes) channels, or sequential 1-s applications of 2 mM MTSET applied at −140 mV on R255N/I262C (black triangles) channels. MTSET was only applied during the inter-episode time at a holding voltage of −100 mV (or −140 mV; as in E). Error bars are SEM. (H and I) Cartoon showing proposed interactions of the positively charged S4 residues (R255, R258, and R261) with the two clusters of negatively charged residues in S1–S3 at the intracellular and extracellular borders of the Hv subunit in I262C (H) and R255N/I262C (I) channels. We propose that it is the interactions of the S4 charges with the two negatively charged clusters that set the limits for the extent of S4 movement (Fig. 3 [A and B] from Gonzalez et al., 2010). Image published in: Gonzalez C et al. (2013) © 2013 Gonzalez et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View

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