Mode shift of the voltage sensors in Shaker K+ channels is caused by energetic coupling to the pore domain

Abstract

The voltage sensors of voltage-gated ion channels undergo a conformational change upon depolarization of the membrane that leads to pore opening. This conformational change can be measured as gating currents and is thought to be transferred to the pore domain via an annealing of the covalent link between voltage sensor and pore (S4-S5 linker) and the C terminus of the pore domain (S6). Upon prolonged depolarizations, the voltage dependence of the charge movement shifts to more hyperpolarized potentials. This mode shift had been linked to C-type inactivation but has recently been suggested to be caused by a relaxation of the voltage sensor itself. In this study, we identified two ShakerIR mutations in the S4-S5 linker (I384N) and S6 (F484G) that, when mutated, completely uncouple voltage sensor movement from pore opening. Using these mutants, we show that the pore transfers energy onto the voltage sensor and that uncoupling the pore from the voltage sensor leads the voltage sensors to be activated at more negative potentials. This uncoupling also eliminates the mode shift occurring during prolonged depolarizations, indicating that the pore influences entry into the mode shift. Using voltage-clamp fluorometry, we identified that the slow conformational change of the S4 previously correlated with the mode shift disappears when uncoupling the pore. The effects can be explained by a mechanical load that is imposed upon the voltage sensors by the pore domain and allosterically modulates its conformation. Mode shift is caused by the stabilization of the open state but leads to a conformational change in the voltage sensor.

Double Mutant Cycle Analysis Identified a Critical Leucine Residue in the IIS4S5 Linker for the Activation of the CaV2.3 Calcium Channel

“…None of the Ca V 2.3 single mutations herein studied were seen to completely uncouple the voltage sensor movement from pore openings, unlike I384N and F484G in homotetrameric Shaker channels (44). This may not be surprising given that our current investigation was limited to domain II.…”

The isolated voltage sensing domain of the Shaker potassium channel forms a voltage-gated cation channel

“…The first four arginines (R1-R4) of S4 sense and interact with the surrounding electric field, and move the S4 outward during depolarization and inward during hyperpolarization in a combination of translation, rotation and tilt (Faure et al, 2012; Li et al, 2014a, 2014b; Vargas et al, 2012; Yarov-Yarovoy et al, 2012). These changes are mechanically transmitted to the pore domain via the S4–S5 linker and the C-terminal S6 of the same and neighboring subunit, leading to gating of the central pore (Batulan et al, 2010; Wall-Lacelle et al, 2011; Haddad and Blunck, 2011; Muroi et al, 2010; Long et al, 2005b; Catterall, 2010; Bezanilla, 2005; Lu et al, 2001, 2002; Labro et al, 2008). Opening of the pore has been shown to occur in at least two steps (Del Camino et al, 2005; Kalstrup and Blunck, 2013).…”

“…Prolonged depolarization has been shown to reconfigure VSDs of numerous voltage-gated proteins into a stable ‘relaxed’ state, resulting in a hyperpolarizing shift of the voltage dependence of both pore closure and return of the VSD to its resting position (Haddad and Blunck, 2011; Piper et al, 2003; Tan et al, 2012; Olcese et al, 1997; Kuzmenkin et al, 2004; Bruening-Wright and Larsson, 2007; Villalba-Galea et al, 2008). Relaxation or mode shift has been shown to be influenced by the state of the pore domain; it was observed to be correlated with slow C-type inactivation (Olcese et al, 1997; Cuello et al, 2010; Männikkö et al, 2005) and pore stabilization in the open state (Haddad and Blunck, 2011).…”

Clinical heterogeneity associated with KCNA1 mutations include cataplexy and nonataxic presentations

“…The T226R mutation has been previously shown to enhance neurotransmitter release without detectable effect on neuronal excitability [15]. Further, the I314 residue has been extensively studied (reported as residue I384 on Shaker gene in drosophila) and has been found to be critically important in coupling of the voltage sensor to pore opening [16]. When residue 314 is mutated from isoleucine to asparagine, it completely uncouples voltage sensor movement from pore opening [16].…”

“…Further, the I314 residue has been extensively studied (reported as residue I384 on Shaker gene in drosophila) and has been found to be critically important in coupling of the voltage sensor to pore opening [16]. When residue 314 is mutated from isoleucine to asparagine, it completely uncouples voltage sensor movement from pore opening [16]. Interestingly, while well conserved across various species and many Kv channels, the Kv5.1 and Kv6.1 channels encoded by KCNF1 and KCNG1 respectively contain a T instead of I at that location, similar to the I314T mutation carried by family 1.…”