Crystal Structure of the Eukaryotic Strong Inward-Rectifier K ⁺ Channel Kir2.2 at 3.1 Å Resolution

Abstract

Inward-rectifier potassium (K + ) channels conduct K + ions most efficiently in one direction, into the cell. Kir2 channels control the resting membrane voltage in many electrically excitable cells and heritable mutations cause periodic paralysis and cardiac arrhythmia. We present the crystal structure of Kir2.2 from chicken, which, excluding the unstructured amino and carboxyl termini, is 90% identical to human Kir2.2. Crystals containing rubidium (Rb + ), strontium (Sr 2+ ), and europium (Eu 3+ ) reveal binding sites along the ion conduction pathway that are both conductive and inhibitory. The sites correlate with extensive electrophysiological data and provide a structural basis for understanding rectification. The channel's extracellular surface, with large structured turrets and an unusual selectivity filter entryway, might explain the relative insensitivity of eukaryotic inward rectifiers to toxins. These same surface features also suggest a possible approach to the development of inhibitory agents specific to each member of the inward-rectifier K + channel family.In 1949 Bernard Katz introduced the term "anomalous rectification" to distinguish the K + currents he observed in frog skeletal muscle from the "delayed rectification" K + currents of the squid axon action potential (1,2). Today we know that "delayed rectifiers" are a subset of the large family of voltage-dependent K + (Kv) channels, whereas "anomalous rectifiers" are members of a different family of channels more commonly known as inward-rectifier K + (Kir) channels (3). The name "inward rectifier" refers to a fundamental ion-conduction property exhibited to a greater or lesser degree by all members of the family: Given an equal but opposite electrochemical driving force, K + conductance into the cell far exceeds conductance out of the cell. Thus, Kir channels are analogous to one-way conductors, or diodes, in solid-state electronic devices.Electrophysiological experiments have shown that inward rectification is a consequence of voltage-dependent pore blockage by intracellular multivalent cations, especially Mg 2+ and polyamines (4-8). At internal negative (hyperpolarizing) membrane voltages the blocking ions are cleared from the pore so that K + conducts, whereas at internal positive (depolarizing) membrane voltages the blocking ions are driven into the pore from the cytoplasm so that K + conduction is blocked. As a result, Kir channels are conductive when an excitable cell is at rest and non-conductive during excitation. This property is thought to foster energy efficiency because it enables Kir channels to regulate the resting membrane potential, but not dissipate the K + gradient during an action potential (3). Another longstanding puzzle in eukaryotic Kir channel studies is their relative insensitivity to natural toxins that typically inhibit other K + channels (22-24). Snake, spider and scorpion venoms, for example, contain numerous toxins against various Kv channels and Ca 2+ -activated K + channels (25)(26)(27). By contrast, Kir ...

Protein interactions central to stabilizing the K + channel selectivity filter in a four-sited configuration for selective K + permeation

“…However, as shown in the structure of Kir2.2, the filter backbone is pinned in a stable four-sited configuration for efficient and selective potassium conduction via a conserved acid-base pair instead of the aromatic to Aspartate hydrogen bond, resulting in a salt bridge again between the pore helix and the external end of the selectivity filter (Fig. 6A, between Glu139 and Arg149) (5). In an effort to recapitulate this structural feature on NaK, we generated mutations to mimic the protein environment surrounding the filter of Kir channels based on sequence and structural comparison between NaK2K and the Kir family (Fig.…”

“…The signature sequence in each subunit adopts an extended structure where all backbone carbonyl groups point toward the central axis; four such elements in the channel tetramer surround the ion conduction pathway, generating four chemically equivalent ion binding sites made up of the backbone carbonyls and the threonine side-chain hydroxyls (2). Although the availability of a number of high-resolution structures of K þ channels has greatly aided our understanding of the molecular details of ion binding in this highly conserved structure (3)(4)(5)(6), the underlying mechanism of K þ selectivity remains elusive (7)(8)(9)(10)(11)(12). The structural studies of K þ channels seem to favor the classical snug-fit model to account for K þ over Na þ selectivity (13,14), while computational studies on K þ channel selectivity invoke a number of different concepts in explaining K þ selectivity (15)(16)(17)(18)(19)(20)(21)(22)(23)(24).…”

“…The role of the bridging hydrogen bond at the extracellular end of the K þ channel filter, equivalent to the Tyr55-Asp68 H bond in NaK2K, was also functionally tested on a eukaryotic voltage-gated K þ channel (rat K v 1.6) (29), which clearly demonstrated that breakage or weakening of this interaction leads to the loss of K þ selectivity and can also result in channel inactivation. In light of the distinct protein interactions stabilizing the filter of inward-rectifier potassium (Kir) channel family as compared to most K þ channels, we also partially recapitulated the Kir channel filter surroundings on NaK and generated a K þ selective NaK mutant that bears the key sequence features of a Kir channel (5,30).…”

Analysis of the selectivity filter of the voltage-gated sodium channel NavRh

“…The pore of KcsA only permits the passage of dehydrated potassium ions [10,11]. This property is highly conserved in most types of potassium channels [4][5][6][12][13][14][15][16][17]. The ion permeation, however, is different in Na v channels where Na + is…”

Domain Organization of the ATP-sensitive Potassium Channel Complex Examined by Fluorescence Resonance Energy Transfer

“…The N termini of Kir channels consist of a helix (termed the slide helix), which lies parallel to the membrane surface and then extends outwards. The large C termini generate a series of ␤-sheets that form a cytoplasmic vestibule, which extends the permeation pathway into the cytoplasm (9,10).…”