Functional Asymmetries and Sidedness of Proton Activation

Basic electrophysiological properties of the KcsA K 1 channel were examined in planar lipid bilayer membranes. The channel displays open-state rectification and weakly voltage-dependent gating. Tetraethylammonium blocking affinity depends on the side of the bilayer to which the blocker is added. Addition of Na 1 to the trans chamber causes block of open-channel current, while addition to the cis side has no effect. Most striking is the activation of KcsA by protons; channel activity is observed only when the trans bilayer chamber is at low pH. To ascertain which side of the channel faces which chamber, residues with structurally known locations were mapped to defined sides of the bilayer. Mutation of Y82, an external residue, results in changes in tetraethylammonium affinity exclusively from the cis side. Channels with cysteine residues substituted at externally exposed Y82 or internally exposed Q119 are functionally modified by methanethiosulfonate reagents from the cis or trans chambers, respectively. Block by charybdotoxin, known to bind to the channel’s external mouth, is observed only when the toxin is added to the cis side of channels mutated to be toxin sensitive. These results demonstrate unambiguously that the protonation sites linked to gating are on the intracellular portion of the KcsA protein. key words: potassium channel • permeation • gating • block I N T R O D U C T I O N The high-resolution structure of the KcsA K 1 channel has invigorated current approaches to the molecular foundations of cellular electrical excitability (Doyle et al., 1998). KcsA is a prokaryotic channel with little sequence similarity to eukaryotic K 1 channels except in the pore-forming region. However, its structure provides compelling explanations for ion permeation and gating phenomena observed over many years in a multitude of K 1 channels. Ironically, functional properties of KcsA have been described only in outline. Singlechannel recording, flux measurements, and ligandbinding assays have shown KcsA to be a high-conductance, tetrameric, K 1 -selective channel with an externally located receptor site for charybdotoxin-family peptides (Schrempf et al., 1995; Cortes and Perozo, 1997; Heginbotham et al., 1997, 1998; MacKinnon et al., 1998). While its structure is largely in harmony with models of familiar K 1 channels, an unexpected characteristic of KcsA is its gating by protons (Cuello et al., 1998; Perozo et al., 1998). The channel reconstituted into planar bilayer membranes opens significantly only at pH values lower than z 5. Despite the fact that most of the protein’s water-exposed mass, with fully 85% of its dissociable protons, is located on the cytoplasmic side of the membrane, proteolysis protection studies have led to the contention that activating protons are sensed on the channel’s external side (Cuello et al., 1998; Perozo et al., 1999). As a prelude to a full ion selectivity study of KcsA, we sought to establish a planar lipid bilayer system in which single purified KcsA channels may be recorded accurately and to survey several basic pore properties of the channel. Single KcsA channels can be observed at 5 kHz bandwidth in a low-noise planar bilayer system. We document functionally asymmetric characteristics of KcsA and use several of these to show that protons gate this channel from the cytoplasmic, not the external, side of the membrane. M A T E R I A L S A N D M E T H O D S