Structure of the full-length Shaker potassium channel Kv1.2 by normal-mode-based X-ray crystallographic refinement

Abstract

Voltage-dependent potassium channels (Kv) are homotetramers composed of four voltage sensors and one pore domain. Because of high-level structural flexibility, the first mammalian Kv structure, Kv1.2 at 2.9 Å, has about 37% molecular mass of the transmembrane portion not resolved. In this study, by applying a novel normalmode-based X-ray crystallographic refinement method to the original diffraction data and structural model, we established the structure of full-length Kv1.2 in its native form. This structure offers mechanistic insights into voltage sensing. Particularly, it shows a hydrophobic layer of about 10 Å at the midpoint of the membrane bilayer, which is likely the molecular basis for the observed "focused electric field" of Kv1.2 between the internal and external solutions. This work also demonstrated the potential of the refinement method in bringing up large chunks of missing densities, thus beneficial to structural refinement of many difficult systems.anisotropic B factors | ion channel | normal-mode refinement | voltage gating V oltage-dependent ion channels sense change in the voltage across cell membrane and respond by allowing specific ions in/out with high selectivity and efficiency (1). Among them, voltage-dependent potassium channels (Kv) have been most extensively studied (2-6). In an action potential, the Kv channel allows intracellular potassium getting out of the cell to return the membrane potential to the polarized state, the failure of which could cause overexcitability of neuron cells, leading to neuronal diseases (7,8). The Kv channel is a homotetramer with four voltage sensors and one central pore domain (9, 10). In each subunit, four transmembrane helices (S1, S2, S3, and S4) make up a voltage-sensor domain, and two transmembrane helices (S5 and S6) contribute to the central pore domain. There are several highly conserved positively charged residues on the S4 helix, the so-called gating charges, which respond to voltage change to open or close the central pore domain (11).The first mammalian Kv crystal structure was of the Shaker family Kv1.2 from Rattus norvegicus [Protein Data Bank (PDB) ID code 2A79], determined to a resolution of 2.9 Å (10). Its structural model was refined by conventional isotropic X-ray crystallographic refinement protocol in CNS (12). Because of the high dynamic and/or static disordering, about 37% of the total molecular mass of the transmembrane portion (residues 131-421) was not resolved. For instance, all the loops connecting S1-S4 helices, and all the side chains on S1 and S3, as well as some of the side chains on S2 and S4, were missing (Fig. 1A). In order to obtain a clearer picture of Kv channels, the structure of a "paddle chimera" Kv was recently determined at a higher resolution of 2.4 Å (13). The improvements in the structure were achieved by crystallizing the channel in a lipid environment and by replacing the voltage-sensor paddle of Kv1.2 with the corresponding region of Kv2.1, a closely related Kv channel in the Shab family. Even though the ch...

Moving gating charges through the gating pore in a Kv channel voltage sensor

“…Because the gating pore residues are mostly hydrophobic across the tree of life, it is generally accepted that their hydrophobicity plays a major role in controlling VSD movement (17,21,38). However, we found that a hydrophobic side chain is only exclusively required for I237, whereas the size of the lateral chain is the critical parameter for S240, F244, and F290.…”

“…The Kv1.2 refined X-ray structure shows that the residue homologous to S240 faces the tip of the side chain of R4 in the depolarized conformation (Fig. 1A) (21). S240 is the only gating pore residue capable of participating in hydrogen bond interactions and is well conserved in the VSD among Kv channels.…”

“…Although the transport of ionized groups through the cell membrane is thermodynamically unfavorable, their transfer is catalyzed in the VSD by the presence of negative counter charges in the S1-S3 segments and water-filled cavities that focus the electric field across a narrow 5-to 10-Å constriction termed the "gating pore," or "hydrophobic plug" (14)(15)(16)(17)(18)(19)(20)(21), positioned at the midlevel of the membrane. Despite its narrow depth, the gating pore effectively insulates the two sides of the lipid bilayer and excludes the permeation of free ions.…”

“…1 A and B) (21,23). Investigations of the VSD hyperpolarized (resting) states using molecular dynamics simulations revealed that the gating pore remains formed by the same cluster of residues upon VSD rearrangements (Fig.…”

Kv Channel S1-S2 Linker Working as a Binding Site of Human β-Defensin 2 for Channel Activation Modulation

“…4). This feature of the hBD2-Kv1.3 interaction interface suggests that the Kv1.3 channel S1-S2 linker likely forms an extended loop similar to the loop in Kv1.2, which was observed by normalmode-based x-ray crystallographic refinement (24) (Fig. 4).…”