Surface structure and its dynamic rearrangements of the KcsA potassium channel upon gating and tetrabutylammonium blocking.
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 alpha-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.
Iwamoto
Masayuki
M
Department of Molecular Physiology and Biophysics, Faculty of Medical Sciences, University of Fukui, Matsuoka, 910-1193 Fukui, Japan.
Shimizu
Hirofumi
H
Inoue
Fumiko
F
Konno
Takashi
T
Sasaki
Yuji C
YC
Oiki
Shigetoshi
S
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating; D026902:Potassium Channel Blockers / Q000494:pharmacology*; D015221:Potassium Channels / Q000737:chemistry* / Q000502:physiology; D024642:Potassium Channels, Voltage-Gated; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D015398:Signal Transduction; D020349:Surface Plasmon Resonance
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C504448:KcsA protein, Streptomyces coelicolor; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D000644:Quaternary Ammonium Compounds; C009405:tetrabutylammonium