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

Abstract

Ion conduction across the cellular membrane requires the simultaneous opening of activation and inactivation gates of the K þ channel pore. The bacterial KcsA channel has served as a powerful system for dissecting the structural changes that are related to four major functional states associated with K þ gating. Yet, the direct observation of the full gating cycle of KcsA has remained structurally elusive, and crystal structures mimicking these gating events require mutations in or stabilization of functionally relevant channel segments. Here, we found that changes in lipid composition strongly increased the KcsA open probability. This enabled us to probe all four major gating states in native-like membranes by combining electrophysiological and solid-state NMR experiments. In contrast to previous crystallographic views, we found that the selectivity filter and turret region, coupled to the surrounding bilayer, were actively involved in channel gating. The increase in overall steady-state open probability was accompanied by a reduction in activation-gate opening, underscoring the important role of the surrounding lipid bilayer in the delicate conformational coupling of the inactivation and activation gates.

Hysteresis of KcsA potassium channel's activation– deactivation gating is caused by structural changes at the channel’s selectivity filter

“…A network of energetically coupled amino acid residues largely mediates the communication between these structural motifs (35). This process is known as "allosteric coupling," and in the pore domain of K + channels is responsible for the functional and structural coupling of the channel's AG and its SF (24,(35)(36)(37)(38)(39)(40). This allosteric coupling underlies a process known as C-type inactivation coupled to activation gating in K + channels (24), and recently we have started to understand this mechanism at the molecular level (31,38,39).…”

Supramolecular Organization and Functional Implications of K+ Channel Clusters in Membranes

“…Afterwards, we washed exactly the same sample in acidic (pH 4) buffers, which is a well-known means to open the activation gate of KcsA. [5,15,16] Unexpectedly, we obtained drastically increased NHHC transfer efficiencies with open channels (Figure 2 A). We confirmed this result by "shuttling" the channels back to the closed state (pH 7), for which the NHHC transfer was again much weaker (Figure 2 B).…”

Supramolekulare Organisation und funktionale Auswirkungen von Ballungen von K+‐Kanälen in Membranen

“…Wir maßen zuerst ein DNP‐ssNMR‐Experiment mit geschlossenen Kanälen (bei pH 7), was die Bildung von Ballungen bestätigte. Danach wuschen wir exakt dieselbe Probenpräparation in saurem Puffer (pH 4), was ein bekanntes Mittel ist, um die Aktivierungspforte von KcsA zu öffnen . Überraschenderweise beobachteten wir drastisch erhöhte Signale in NHHC‐Experimenten mit geöffneten Kanälen (Abbildung A).…”