Voltage-Gated Potassium Channels: A Structural Examination of Selectivity and Gating

Abstract

Voltage-gated potassium channels play a fundamental role in the generation and propagation of the action potential. The discovery of these channels began with predictions made by early pioneers, and has culminated in their extensive functional and structural characterization by electrophysiological, spectroscopic, and crystallographic studies. With the aid of a variety of crystal structures of these channels, a highly detailed picture emerges of how the voltage-sensing domain reports changes in the membrane electric field and couples this to conformational changes in the activation gate. In addition, high-resolution structural and functional studies of K(+) channel pores, such as KcsA and MthK, offer a comprehensive picture on how selectivity is achieved in K(+) channels. Here, we illustrate the remarkable features of voltage-gated potassium channels and explain the mechanisms used by these machines with experimental data.

Figure 1.

Structural details of KcsA. (A) KcsA tetramer as viewed from the top of the membrane (PDBID:14KC). Each subunit is uniquely colored. The central ion conduction pore is shown with a K⁺ ion (blue sphere). (B) KcsA as viewed from the side with two opposing subunits removed for clarity. The P-loop contains the signature sequence TVGYG (shown as sticks), and forms the selectivity filter. Four potassium ions are shown in the selectivity filter. Below the selectivity filter is the aqueous cavity, formed by the TM2 helices that form the bundle crossing. (C) A detailed view of the selectivity filter of KcsA. Each binding site, S0–S4, is formed by the oxygen cages originating from backbone carbonyl and side-chain hydroxyls of the selectivity filter signature sequence TVGYG. Dashed lines depict coordination of the K⁺ ion in S4 and the oxygens. Sodium (orange sphere) binds in the plane between sites S3 and S4. Dashed lines represent its coodination with carbonyl oxygens.

Figure 2.

Gating mechanisms of potassium channels. (A) Gating can occur via conformational changes at the bundle crossing. The inner helices physically block the entry of potassium ions into the aqueous cavity, as shown in the KcsA closed structure (left structure, 1K4C). On activation, the inner helices splay open as observed in MthK (right structure, 1LNQ) and expose the cavity. Blue spheres denote potassium ions. (B) C-type inactivation and selectivity filter gating. In this model, partial collapse of the filter prevents conduction of potassium ions through the pore even if the bundle-crossing gate is open. This model is based on the comparison of the open MthK structure (left structure, 1LNQ) to the collapsed filter of a mutant KcsA in the open state (right structure, 3F5W). (C) N-type inactivation, or ball-and-chain gating, results from binding of an auto-inhibitory peptide to the bundle-crossing gate to physically block entry of ions into the cavity. This peptide can be part of the amino terminus of the channel (as shown) or the amino terminus of an associated β subunit. Currently, no crystal structures exist to illustrate N-type inactivation. The left panels show a cartoon representation of the gating mechanism, and the right panels show the available crystal structures that represent these conformational states.

Figure 3.

Structure of the voltage-dependent potassium channel. (A) Membrane topology of Kv channels consists of six transmembrane helices S1–S6, where both the amino and carboxyl termini are intracellular. Helices S1–S4 form the voltage sensor, whereas helices S5 and S6 comprise the channel pore and are analogous to TM1 and TM2 of KcsA and MthK. The S4 helix contains the conserved positively charged residues (plus signs) that are critical to the voltage-sensing mechanism. (B) The crystal structure of a chimeric channel comprised of the Kv1.2 with the voltage-sensor paddle of Kv2.1, crystallized in the presence of detergent and lipids (PDBID:2R9R). The β subunit is bound to the channel via the T1 intracellular domain. One subunit is depicted here and helices S1–S6, the S4–S5 linker, the PVP hinge, and the T1–S1 linker are indicated. (C) Side view of the channel tetramer β subunit complex. (D) Top view of the channel tetramer. Each subunit is differently colored, and the channel pore is visible in the center of the structure. The voltage-sensing domains are located outside of the channel pore. A potassium ion is shown as a blue sphere. (E) A detailed view of the voltage-sensor domain of the chimeric channel. Critical arginine residues are shown as green sticks and labeled as the putative gating charges R0–R4, followed by K5 and R6 on the S4 helix. Two clusters of negatively charged residues serve as countercharges to the gating charges on the external side (E183 and E226) and the internal side (E154, E236, and D259) and are separated by the F233 on S2. (F) The 1.9 Å resolution structure of the isolated voltage-sensor domain of KvAP (PDBID:1ORQ). Critical arginine residues on the S4 helix are shown in green sticks. Gating charge R133 forms a salt bridge with D62 on the S2 helix, whereas R76, D72, and E93 form a salt-bridge network to bridge S2 with S3a.

Figure 4.

A model for coupling of voltage sensing to channel opening in Kv channels. (A) A cartoon depiction of the paddle model (Long et al. 2007) voltage-sensing mechanism. Helices S1–S4 and the S4–S5 linker are shown as cylinders of different colors. Gating charges are located on the S4 helix and represented by a black + sign. Countercharges on S0 (a short α-helical region before S1), S1, and S2 are represented by a red dash. In the up or open conformation (left), S3 and S4 are located within the membrane and the gating charges are closer to the extracellular side, interacting with the external cluster of countercharges. The hydrophobic plug is represented by an orange circle on the S2 helix. A change in membrane potential would cause the S4 helix to move into the down conformation (right) with the gating charges now closer to the intracellular side and interacting with the internal cluster of countercharges. This displacement of S4 pushes down on the S4–S5 linker, tilting it toward the intracellular side, poising it to interact with the S6 helices to close the pore. (B) The proposed mechanism for coupling of voltage sensing to gating in Kv channels (Long et al. 2007). The channel pore and bundle crossing are represented in blue. S4 and the S4–S5 helix of the voltage-sensing domain are depicted in green and red, respectively. Based on the crystal structure, S4 is in the up conformation (left), and the S4–S5 linker rests on the S6 helices in the bundle crossing in the open state. Transition to the hypothetical closed state of the channel requires movement of S4 into the down conformation (right). This downward movement of the S4 helix pushes on the amino-terminal end of the S4–S5 helix, which tilts toward the intracellular side and pushes the S6 helices down into the closed state. (C) A comparison of the conformational states of the voltage-sensing phosphatase Ci-VSP. R217E Ci-VSP (left, PDBID:4G7V) shows the voltage sensor in the up conformation, whereas wild-type Ci-VSP (right, PDBID:4G80) shows the voltage sensor in the down conformation. The structures were aligned using the S1 helix for reference. Critical Arg residues R1–R4 are shown as green sticks. Countercharges are shown along with residues comprising the hydrophobic plug region. The gray dotted lines denote the position of R1 (top) and R4 (bottom) on the up conformation of R217E Ci-VSP for comparison to their positions on the down conformation of wild-type.