Structural basis for the coupling between activation and inactivation gates in K+ channels

Abstract

The coupled interplay between activation and inactivation gating is a functional hallmark of K+ channels1,2. This coupling has been experimentally demonstrated from ion interaction effects3,4, cysteine accessibility1 and is associated with a well-defined boundary of energetically coupled residues2. The structure of KcsA in its fully open conformation, as well as four other partial openings, richly illustrates the structural basis of activation-inactivation gating5. Here, we have identified the mechanistic principles by which movements on the inner bundle gate trigger conformational changes at the selectivity filter, leading to the non-conductive C-type inactivated state. Analysis of a series of KcsA open structures suggests that as a consequence of the hinge bending and rotation of TM2, the aromatic ring of Phe103 tilts towards residues Thr74 and Thr75 in the pore helix as well as Ile100 in the neighboring subunit. This allows the network of hydrogen bonds among residues W67, E71, and D80 to destabilize the selectivity filter6,7, facilitating entry to its non-conductive conformation. Mutations at position 103, affect gating kinetics in a size-dependent way: small side chain substitutions F103A and F103C severely impair inactivation kinetics, while larger side chains (F103W) have more subtle effects. This suggests that the allosteric coupling between the inner helical bundle and the selectivity filter might rely on straightforward mechanical deformation propagated through a network of steric contacts. Average interactions calculated from molecular dynamics simulations show favourable open state interaction-energies between Phe103 and surrounding residues. Similar interactions were probed in the Shaker K-channel where inactivation was impaired in the mutant I470A. We propose that side chain rearrangements at position 103 mechanically couple activation and inactivation in KcsA and a variety of other K channels.

Probing Conformational Changes during the Gating Cycle of a Potassium Channel in Lipid Bilayers

“…This enabled us to study for the first time, to our knowledge, the structure of a functionally defined open state of the KcsA channel. A comparison of our findings for wild-type (WT) KcsA with earlier crystallographic results for KcsA mutants (13,14,25) led to a detailed understanding of the conformational changes that occur during the KcsA gating cycle in lipid bilayers. The data provide a general picture of how ion channels transit from activated to inactivated states and back.…”

“…SsNMR experiments were conducted on NMR spectrometers operating at 500 and 700 MHz 1 H resonance frequencies (Bruker Biospin, Germany) equipped with 3.2 mm triple-resonance ( 1 H, 13 C, 15 N) magic-angle spinning (MAS) probe heads (see, e.g., (26)). All experiments were carried out at an effective sample temperature of 273 K using MAS frequencies of 11.4, 12, or 15 kHz.…”

“…All experiments were carried out at an effective sample temperature of 273 K using MAS frequencies of 11.4, 12, or 15 kHz. 13 C-13 C proton-driven spin diffusion (PDSD) experiments used homonuclear mixing times of 30 ms and 150 ms. The latter experiments were conducted under weak coupling conditions (27).…”

“…The resting state of the KcsA channel, with the inactivation gate open and the activation gate closed (closed-conductive state), and the inactivated state, with the inactivation gate closed and the activation gate open (open-inactivated state), are relatively long-lived. Therefore, conformational changes associated with the opening and closing of the KcsA activation and inactivation gates can be studied with the use of crystallographic (10,11,13,14) and spectroscopic methods. The inner KcsA helices form an important part of the conduction pathway.…”

“…Despite these advancements, a direct structural visualization of the entire KcsA gating cycle, including the conformation with an open activation and closed inactivation gate (open-conductive conformation) in a lipidic environment has remained challenging due to the very short lifetime of this channel state and the propensity of the selectivity filter to rapidly collapse when the activation gate is opened (12)(13)(14)24,25). Using solid-state NMR spectroscopy, we previously identified a lipid-KcsA interaction site at the turret region, which connects the outer transmembrane helix of KcsA (transmembrane helix 1 (TM1)) and the pore helix behind the selectivity filter of the KcsA channel.…”