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...

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