Études de type structure fonction du couplage électromécanique et de la coopérativité sous-unitaire chez les canaux potassiques dépendants du voltage
Voltage-gated potassium channels are tetramers and each subunit is formed of six transmembrane segments (S1 to S6). The pore, formed by the S5-S6 segments of each subunit, is surrounded by four modules responsible for sensitivity to the membrane potential, the voltage sensors (VS, S1-S4). During membrane depolarization, the movement of charged residues located in the VS causes a detectable charge movement called the gating current. The activation of the VS led to the opening of the pore, resulting in a conformational change in the C-terminal segment of S6. To elucidate the principles underlying the electromechanical coupling between these two domains, we examined two regions presumed responsible for the coupling among channels of the Shaker K + family: the carboxy-terminal region of S6 and the peptide bond linking the transmembrane segments S4-S5 (S4-5L). Using the cut-open voltage clamp fluorometry (COVCF), we have determined that the RELY inter-subunit interaction, formed by amino acids located on the S4-5L linker and S6 of two neighboring subunits, is involved in the development of the slow component observed during the return of the gating charges (OFF-gating) to their resting state. The introduction of mutations in the RELY region modulates the strength of these molecular interactions and eliminates the asymmetry observed in the wild type gating currents. Moreover, we demonstrate that this inter-subunit coupling is responsible for stabilizing the pore in the open state. We have also identified an intra-subunit interaction between residues I384 located on the S4-5L linker and F484 on the S6 segment of the same subunit. The destabilization of this hydrophobic interaction uncouples completely the movement of voltage sensors from pore opening. Without this interaction, the energy required to activate the VS is diminished due to the absence of mechanical weight applied by the pore. Furthermore, this  uncoupling also eliminates the "mode shift", defined as an amplified shift of the voltage dependence of gating charge (QV) to hyperpolarizing potentials during prolonged depolarization, thus indicating that the mechanical load of the pore influences the entry of the VS into this shifted mode  by modulating the conformation of the VS threw an intrinsic allosteric process.
Potassium channels Electromecanical coupling Mode-Shift Mode-shift Courant de gating Gating currents Cooperativity Fluorometry Voltage clamp