The bacterial potassium channel KcsA is gated by high concentrations of intracellular protons, allowing the channel to open at pH < 5.5. Despite prior attempts to determine the mechanism responsible for pH gating, the proton sensor has remained elusive. We have constructed a KcsA channel mutant that remains open up to pH 9.0 by replacing key ionizable residues from the N and C termini of KcsA with residues mimicking their protonated counterparts with respect to charge. A series of individual and combined mutations were investigated by using single-channel recordings in lipid bilayers. We propose that these residues are the protonbinding sites and at neutral pH they form a complex network of inter-and intrasubunit salt bridges and hydrogen bonds near the bundle crossing that greatly stabilize the closed state. In our model, these residues change their ionization state at acidic pH, thereby disrupting this network, modifying the electrostatic landscape near the channel gate, and favoring channel opening.ion channel ͉ proton sensor ͉ salt bridge network ͉ pH gating A ctivity of ion channel proteins is modulated by signaling molecules that tightly control the opening and closing of the channel pores, allowing ions to cross the membrane in response to cellular signals. Protons are ubiquitous modulators of ion channel gating and permeation, likely because of the presence of titratable residues located near channel gates, pores, allosteric sites, and regulatory interfaces. Ion channels sensitive to either cytoplasmic or extracellular pH include: transient receptor potential (TRP) and acid-sensing (ASIC) channels, inward rectifier potassium channels (Kir), CLC chloride channels, NMDA receptors, and Ca-activated potassium channels (1-7). Strict modulation of channel gating near neutral pH is often crucial for the physiological roles of these channels. Despite the importance of pH modulation in these channels, the molecular mechanisms of proton gating are not completely understood, partly because of the absence of detailed structural information.The prokaryotic potassium channel KcsA, the first K ϩ channel characterized with x-ray crystallography (8), is modulated by pH in a very narrow acidic pH range (9, 10). The availability of an atomic structure combined with a sensitive functional assay (electrophysiological current recordings with purified channel protein) make KcsA an ideal system for locating and dissecting its pH sensor. KcsA senses pH at its intracellular side (10) and the pH sensor location has been further narrowed by truncation constructs that maintain the pH sensitivity of the full-length channel (11). Moreover, an NMR study recently implicated a histidine located near the bundle crossing of KcsA as the pH sensor (12). Despite major advances in our understanding of this archetypal ion channel, there is no detailed molecular picture of the pH sensor. Elucidating the mechanism underlying KcsA pH sensing may provide a foundation for understanding similar pH-gating dependencies in eukaryotic potassium channels....

Voltage-gated ion channels open and close in response to voltage changes across electrically excitable cell membranes. Voltage-gated potassium (Kv) channels are homotetramers with each subunit constructed from six transmembrane segments, S1-S6 (ref. 2). The voltage-sensing domain (segments S1-S4) contains charged arginine residues on S4 that move across the membrane electric field, modulating channel open probability. Understanding the physical movements of this voltage sensor is of fundamental importance and is the subject of controversy. Recently, the crystal structure of the KvAP channel motivated an unconventional 'paddle model' of S4 charge movement, indicating that the segments S3b and S4 might move as a unit through the lipid bilayer with a large (15-20-A) transmembrane displacement. Here we show that the voltage-sensor segments do not undergo significant transmembrane translation. We tested the movement of these segments in functional Shaker K+ channels by using luminescence resonance energy transfer to measure distances between the voltage sensors and a pore-bound scorpion toxin. Our results are consistent with a 2-A vertical displacement of S4, not the large excursion predicted by the paddle model. This small movement supports an alternative model in which the protein shapes the electric field profile, focusing it across a narrow region of S4 (ref. 6).

Understanding how ion channels open and close their pores is crucial for understanding their physiological roles. We used intracellular quaternary ammonium blockers to locate the voltage-dependent gate in MthK potassium channels from Methanobacterium thermoautotrophicum with electrophysiology and X-ray crystallography. Blockers bind in an aqueous cavity between two putative gates, an intracellular gate and the selectivity filter. Thus, these blockers directly probe gate location: an intracellular gate will prevent binding when closed, whereas a selectivity filter gate will always allow binding. A kinetic analysis of tetrabutylammonium block of single MthK channels combined with X-ray crystallographic analysis of the pore with tetrabutylantimony unequivocally determined that the voltage-dependent gate, like the C-type inactivation gate in eukaryotic channels, is located at the selectivity filter. State-dependent binding kinetics suggests that MthK inactivation leads to conformational changes within the cavity and intracellular pore entrance.

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Potassium channels are opened by ligands and/or membrane potential. In voltage-gated K+ channels and the prokaryotic KcsA channel, conduction is believed to result from opening of an intracellular constriction that prevents ion entry into the pore. On the other hand, numerous ligand-gated K+ channels lack such gate, suggesting that they may be activated by a change within the selectivity filter, a narrow region at the extracellular side of the pore. Using molecular dynamics simulations and electrophysiology measurements, we show that ligand-induced conformational changes in the KcsA channel removes steric restraints at the selectivity filter, thus resulting in structural fluctuations, reduced K+ affinity, and increased ion permeation. Such activation of the selectivity filter may be a universal gating mechanism within K+ channels. The occlusion of the pore at the level of the intracellular gate appears to be secondary.