Surface Structure and Its Dynamic Rearrangements of the KcsA Potassium Channel upon Gating and Tetrabutylammonium Blocking

Abstract

KcsA is the first potassium channel for which the molecular structure was revealed. However, the high resolution structural information is limited to the transmembrane domain, and the dynamic picture of the full KcsA channel remains unsolved. We have developed a new approach to investigate the surface structure of proteins, and we applied this method to investigate the full length of the KcsA channel. Single-cysteine substitution was introduced into 25 sites, and specific reaction of these mutated channels to a bare surface of a flat gold plate was evaluated by surface plasmon resonance measurements. The surface plasmon resonance signals revealed the highest exposure for the mutant of the C-terminal end. When the gate of the KcsA channel is kept closed at pH 7.5, the extent of exposure showed periodic patterns for the consecutive sites located in the cytoplasmic (CP) and N-terminal domain. This suggests that these stretches take the ␣-helical structure. When the channel was actively gated at pH 4.0, many sites in the CP domain became exposed. Compared with the rigid structure in pH 7.5, these results indicate that the CP domain became loosely packed upon active gating. The C-terminal end of the M2 helix is a moving part of the gate, and it is exposed to the outer surface slightly at pH 4.0. By adding a channel blocker, tetrabutylammonium, the gate is further exposed. This suggests that in the active gating tetrabutylammonium keeps the gate open rather than being trapped in the central cavity.Conformational changes of a protein rearrange the surface structure. Some residues become exposed, whereas others are enclosed in the interior. Thus the detection of changes in the exposure of residues will give information on the underlying conformational changes. Various methods, such as the substituted cysteine accessibility method (1-4), and various types of probe molecules have been used to reveal the geometry of the surface for various proteins. When a probe binds to the reactive site on the protein, the reactivity depends strongly on the size of the probe. Small probes detect whether an amino acid residue is either exposed or buried. Intermediate probes explore cavities in proteins. Large probes outline the outer surfaces of proteins. What would happen if the size of the probe becomes infinite? A probe cannot access the protein unless the target sites protrude. Following this idea we developed a simple approach. As a "probe" with an infinite size, a bare flat gold piece was used, and reactions of cysteine-mutated proteins to the gold plate were detected by surface plasmon resonance (SPR) 2 signals. This method should provide information about the rough surface structure of proteins.In ion channels, domains extending out from the transmembrane domain (TM domain), either to the extracellular or intracellular space, pick up chemical signals and transfer information to the TM domain (5). In addition to this role as sensor, the cytoplasmic domains (CP domains) are known to regulate the assembly of the oligomeric structur...

Functional Equilibrium of the KcsA Structure Revealed by NMR

“…The bath solutions were made asymmetric in terms of pH (pH 7.5 for the cis side, pH 4.0 for the trans side). Because one of the two gates, the helix bundle crossing on the intracellular side opens when the intracellular pH is acidic, the gate of the channels oriented with the intracellular region facing the trans side is constitutively open, and the gate of the channels oppositely oriented is closed (22). Therefore, the currents observed here are caused by opening of another gate, the selectivity filter, of the channels oriented with the intracellular region facing the trans side.…”

“…KcsA was reconstituted into liposomes as described previously (22). Two chambers (cis and trans), divided with a plate with a hole (50 -100 m in diameter), were used for the analyses.…”

“…5A, top panel) (22). When equal amounts of TFE were added to the cis and trans sides to a final concentration of 3%, by 0.5 per step, the channel gradually increased its activity (Fig.…”

Amphipathic antenna of an inward rectifier K + channel responds to changes in the inner membrane leaflet

“…A planar lipid bilayer method was used for the single-channel current measurements, as reported previously (42). A Teflon sheet of 0.5-mm thickness, with an aperture of <100-μm diameter was used as a partition for the two compartments (cis and trans) of a chamber.…”

“…The expression, purification, and reconstitution into liposomes of the wild-type and mutant KcsA channels were described previously (42). The proteoliposomes were made with a protein/lipid weight ratio of 1:2,000 and were passed through a Bio-Spin30 column (Bio-Rad) equilibrated with buffer solution (200 mM KCl, 10 mM Hepes, pH 7.5).…”