Molecular properties of Kcv, a virus encoded K+ channel.
The miniature viral K+ channel Kcv represents the pore module of all K+ channels. A synthetic gene of Kcv with an elevated GC content compared to that of the wild-type gene was expressed heterologously in Pichia pastoris, and the purified protein was functionally reconstituted into liposomes. Biochemical assays reveal a remarkable cation selective stability of the channel tetramer via SDS-PAGE. Only cations, which permeate Kcv, were able to protect the oligomer against disassembly into monomers at high temperatures. Electrophysiological characterization of the single Kcv channel reveals a saturating conductance (lambda(max)) of 360 pS; the single-channel current-voltage relation was strongly rectifying with a negative slope conductance at extreme voltages. The channel was highly selective for K+ and was blocked by Ba2+ and in a side specific manner by Na+ and Cs+ also. The channel conducted Rb+, but as a consequence, the channel was shifted into a hyperactive state. We conclude that specific binding interactions of cations in the conductive pathway are an important determinant of channel stability and function.
Pagliuca
Cinzia
C
Dipartimento di Biologia e IBF-CNR, Universitá degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy.
Goetze
Tom Alexander
TA
Wagner
Richard
R
Thiel
Gerhard
G
Moroni
Anna
A
Parcej
David
D
D000595:Amino Acid Sequence; D001464:Barium / Q000494:pharmacology; D001483:Base Sequence; D002414:Cations, Monovalent / Q000494:pharmacology; D002586:Cesium / Q000494:pharmacology; D004279:DNA, Viral / Q000235:genetics; D004553:Electric Conductivity; D015870:Gene Expression; D005814:Genes, Viral; D008969:Molecular Sequence Data; D017862:Phycodnaviridae / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics; D026902:Potassium Channel Blockers / Q000494:pharmacology; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D002414:Cations, Monovalent; D004279:DNA, Viral; C405463:Kcv potassium channel, Chlorella virus; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D011994:Recombinant Proteins; D014764:Viral Proteins; D002586:Cesium; D001464:Barium