Residues Connecting Voltage Sensor Domain to Pore Domain in Shaker K+ Channel by Noncanonical Coupling Mechanism

In voltage-gated K+ channels (Kv), membrane depolarization promotes a structural reorganization of each of the four voltage sensor domains surrounding the conducting pore, inducing its opening. Although the crystal structure of Kv1.2 provided the first atomic resolution view of a eukaryotic Kv channel, several components of the voltage sensors remain poorly resolved. In particular, the position and orientation of the charged arginine side chains in the S4 transmembrane segments remain controversial. Here we investigate the proximity of S4 and the pore domain in functional Kv1.2 channels in a native membrane environment using electrophysiological analysis of intersubunit histidine metallic bridges formed between the first arginine of S4 (R294) and residues A351 or D352 of the pore domain. We show that histidine pairs are able to bind Zn2+ or Cd2+ with high affinity, demonstrating their close physical proximity. The results of molecular dynamics simulations, consistent with electrophysiological data, indicate that the position of the S4 helix in the functional open-activated state could be shifted by ∼7–8 Å and rotated counterclockwise by 37° along its main axis relative to its position observed in the Kv1.2 x-ray structure. A structural model is provided for this conformation. The results further highlight the dynamic and flexible nature of the voltage sensor.

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Molecular Movement of the Voltage Sensor in a K Channel

The Journal of General Physiology ◽

10.1085/jgp.200308927 ◽

2003 ◽

Vol 122 (6) ◽

pp. 741-748 ◽

Cited By ~ 55

Author(s):

Amir Broomand ◽

Roope Männikkö ◽

H. Peter Larsson ◽

Fredrik Elinder

Keyword(s):

Voltage Sensor ◽

Disulphide Bond ◽

K Channel ◽

Crystallographic Structure ◽

X Ray ◽

Voltage Gated ◽

Disulphide Bonds ◽

State Dependent ◽

Molecular Movement ◽

Pore Domain

The X-ray crystallographic structure of KvAP, a voltage-gated bacterial K channel, was recently published. However, the position and the molecular movement of the voltage sensor, S4, are still controversial. For example, in the crystallographic structure, S4 is located far away (>30 Å) from the pore domain, whereas electrostatic experiments have suggested that S4 is located close (<8 Å) to the pore domain in open channels. To test the proposed location and motion of S4 relative to the pore domain, we induced disulphide bonds between pairs of introduced cysteines: one in S4 and one in the pore domain. Several residues in S4 formed a state-dependent disulphide bond with a residue in the pore domain. Our data suggest that S4 is located close to the pore domain in a neighboring subunit. Our data also place constraints on possible models for S4 movement and are not compatible with a recently proposed KvAP model.

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Molecular basis for functional connectivity between the voltage sensor and the selectivity filter gate in Shaker K+ channels

eLife ◽

10.7554/elife.63077 ◽

2021 ◽

Vol 10 ◽

Author(s):

Carlos AZ Bassetto ◽

João Luis Carvalho-de-Souza ◽

Francisco Bezanilla

Keyword(s):

Molecular Basis ◽

Ionic Conduction ◽

Voltage Sensor ◽

Selectivity Filter ◽

Coupling Mechanism ◽

Charge Voltage ◽

Conductive Channel ◽

Voltage Gated Channels ◽

A Chain ◽

Pore Domain

In Shaker K+ channels, the S4-S5 linker couples the voltage sensor (VSD) and pore domain (PD). Another coupling mechanism is revealed using two W434F-containing channels: L361R:W434F and L366H:W434F. In L361R:W434F, W434F affects the L361R VSD seen as a shallower charge-voltage (Q-V) curve that crosses the conductance-voltage (G-V) curve. In L366H:W434F, L366H relieves the W434F effect converting a non-conductive channel in a conductive one. We report a chain of residues connecting the VSD (S4) to the selectivity filter (SF) in the PD of an adjacent subunit as the molecular basis for voltage sensor selectivity filter gate (VS-SF) coupling. Single alanine substitutions in this region (L409A, S411A, S412A, or F433A) are enough to disrupt the VS-SF coupling, shown by the absence of Q-V and G-V crossing in L361R:W434F mutant and by the lack of ionic conduction in the L366H:W434F mutant. This residue chain defines a new coupling between the VSD and the PD in voltage-gated channels.

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