Importance of lipid–pore loop interface for potassium channel structure and function

Abstract

Potassium (i.e., K + ) channels allow for the controlled and selective passage of potassium ions across the plasma membrane via a conserved pore domain. In voltage-gated K + channels, gating is the result of the coordinated action of two coupled gates: an activation gate at the intracellular entrance of the pore and an inactivation gate at the selectivity filter. By using solid-state NMR structural studies, in combination with electrophysiological experiments and molecular dynamics simulations, we show that the turret region connecting the outer transmembrane helix (transmembrane helix 1) and the pore helix behind the selectivity filter contributes to K + channel inactivation and exhibits a remarkable structural plasticity that correlates to K + channel inactivation. The transmembrane helix 1 unwinds when the K + channel enters the inactivated state and rewinds during the transition to the closed state. In addition to wellcharacterized changes at the K + ion coordination sites, this process is accompanied by conformational changes within the turret region and the pore helix. Further spectroscopic and computational results show that the same channel domain is critically involved in establishing functional contacts between pore domain and the cellular membrane. Taken together, our results suggest that the interaction between the K + channel turret region and the lipid bilayer exerts an important influence on the selective passage of potassium ions via the K + channel pore. membrane protein | ion channel | solid-state NMR spectroscopy P otassium (i.e., K + ) channels are embedded in the plasma membrane to control the selective passage of potassium ions across the lipid bilayer. The channels open and close their conduction pathway by sensing changes in physicochemical parameters such as pH, ligand concentration, and membrane voltage (1). Structure-function studies on voltage-gated K + (Kv) channels suggested that lipid molecules are an integral part of the voltage-sensing domains, which transfer during the gating process electrical charges across the cell membrane (2-4). In some of the available Kv channel crystal structures, lipid molecules appear most densely packed against the pore domain, presumably providing an appropriate environment for the stability and the operation of the gating machinery to open and close the conduction pathway. In general, the activity of Kv pore domains is thought to be determined by the activity of two gates in series, one for activation and one for inactivation. These gates jointly control the conduction of ions through the pore (5-11). The activation gate is located at the intracellular entrance of the pore and the inactivation gate is situated toward the extracellular entrance at the selectivity filter (i.e., C-type inactivation). In addition, some potassium channels possess close to the activation gate a receptor for an N-terminal inactivating domain (i.e., N-type inactivation).The K + channel pore domain is conserved across all K + channels. It comprises a tetrameric assembly of t...

1H‐Detected Solid‐State NMR Studies of Water‐Inaccessible Proteins In Vitro and In Situ

“…We could assign approximately half of the resonances in the spectrum in Figure 2 B based on 3D CαNH, Cα(CO)NH, CONH, and CO(Cα)NH experiments (Figure 2 F), supplemented with 3D NCαCX and 2D N(CO)CX spectra to identify residue types, and supported by available 13 C and 15 N chemical‐shift data 9c, 12. These assignments include a number of functionally essential structural elements that we can access here for the first time with 1 H detection.…”

“…This suggests that the dynamics of the filter are changed in cellular KcsA, which is further corroborated by a relatively strong 15 N CSP of +0.8 ppm for T74 in situ (Figures 3 F and S11). A potential reason for this apparently different behavior of the filter may be the altered lipid composition in the bacterial membrane, which is known to influence the filter,9c or differential clustering of KcsA channels 20. Against the backdrops presented here, it is of great importance to explore how molecular motion modulates channel function.…”

“…The outer membrane was removed to increase the amount of KcsA in the sample, which was then incubated in deuterated solvent for three days prior to the ssNMR experiments. We obtained a surprisingly well‐resolved in situ 2D NH spectrum (Figure 2 E), which exhibited the clear spectral fingerprint of closed‐conductive KcsA9c without any discernible background signal (Figure S6). This is remarkable as the KcsA concentration in situ was about seven times lower than in vitro (see Section S10 in the Supporting Information).…”

“…Whereas only the exchangeable Y78 and G79 residues were detectable in a former study,2f here we could also assign residues T74–G77, providing access to the entire filter. Moreover, we assigned the pore helix L66–E71, a critical element for C‐type inactivation,9a as well as large stretches of the TM1 (transmembrane 1) helix including residues L41–L49, which precede the lipid‐sensitive turret 9c. These signals could be largely retrieved in cellular KcsA.…”

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

“…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. The interaction between the turret and lipid modulates the conduction properties of the KcsA channel pore (4,26). Here, we exploited that earlier observation and modulated KcsA channel gating by varying the lipid composition of the surrounding bilayer.…”