Berneche, S. | Search bioRxiv

bioRxiv2019

The voltage-sensing mechanism of the KvAP channel involves breaking of the S4 helix

Voltage-gated ion channels allow ion permeation upon changes of the membrane electrostatic potential (Vm). Each subunit of these tetrameric channels is composed of six transmembrane helices, of which the anti-parallel helix bundle S1-S4 constitutes the voltage-sensor domain (VSD) and S5-S6 forms the pore domain. Here, using molecular dynamics (MD) simulations, we report novel responses of the archaebacterial potassium channel KvAP to cell polarization. We show that the S4 helix, which is straight in the experimental crystal structure solved under depolarized conditions (Vm [~] 0), breaks into two segments when the cell is polarized (Vm << 0), and reversibly forms a single straight helix following depolarization of the cell (Vm =0). The outermost segment of S4 translates along the normal to the membrane, bringing new perspective to previously paradoxical accessibility experiments that were initially thought to imply the displacement of the whole VSD across the membrane. Our simulations of KvAP reveal that the breaking of S4 under polarization is not a feature unique to hyperpolarization activated channel, as might be suggested by recent cryo-EM structures and MD simulations of the HCN channel.

bioinformatics↗

bioRxiv2019

Divergent Cl-and H + pathways underlie transport coupling and gating in CLC exchangers and channels

The CLC family of anion transporting proteins is comprised of secondary active H+-coupled exchangers and of Cl- channels. Both functional subtypes play key roles in human physiology, and mutations causing their dysfunction lead to numerous genetic disorders. Current models suggest that the CLC exchangers do not utilize a classical ping-pong mechanism of antiport, where the transporter sequentially interacts with one substrate at a time. Rather, in the CLC exchangers both substrates bind and translocate simultaneously while moving through partially congruent pathways. How ions of opposite electrical charge bypass each other while moving in opposite directions through a shared permeation pathway remains unknown. Here, we use MD simulations in combination with biochemical and electrophysiological measurements to identify a pair of highly conserved phenylalanine residues that form an aromatic pathway, separate from the Cl- pore, whose dynamic rearrangements enable H+ movement. Mutations of these aromatic residues impair H+ transport and voltage-dependent gating in the CLC exchangers. Remarkably, the role of the aromatic pathway is evolutionarily conserved in CLC channels. Using atomic-scale mutagenesis we show that the electrostatic properties and conformational flexibility of these aromatic residues are essential determinants of channel gating. Our results suggest that Cl- and H+ move through physically distinct and evolutionarily conserved routes through the CLC channels and transporters. We propose a unifying mechanism that describes the gating mechanism of CLC exchangers and channels.

biochemistry↗