Long Distance Interactions within the Potassium Channel Pore Are Revealed by Molecular Diversity of Viral Proteins

Sabrina Gazzarrini,Ming Kang,James L Van Etten,Sascha Tayefeh,Stefan M Kast,Dario Difrancesco,Gerhard Thiel,Anna Moroni

doi:10.1074/jbc.m401184200

Abstract

Kcv is a 94-amino acid protein encoded by chlorella virus PBCV-1 that corresponds to the pore module of K(+) channels. Therefore, Kcv can be a model for studying the protein design of K(+) channel pores. We analyzed the molecular diversity generated by approximately 1 billion years of evolution on kcv genes isolated from 40 additional chlorella viruses. Because the channel is apparently required for virus replication, the Kcv variants are all functional and contain multiple and dispersed substitutions that represent a repertoire of allowed sets of amino acid substitutions (from 4 to 12 amino acids). Correlations between amino acid substitutions and the new properties displayed by these channels guided site-directed mutations that revealed synergistic amino acid interactions within the protein as well as previously unknown interactions between distant channel domains. The effects of these multiple changes were not predictable from a priori structural knowledge of the channel pore.

Highlights

Steady-state currents measured at Ϫ100 mV in 50 mM Rbϩ or 50 mM Kϩ plus 10 mM Csϩ solutions, are expressed as ratios of current measured in 50 mM Kϩ only
We took advantage of the natural diversity of a family of Kcv channels encoded by chlorella viruses to identify amino acid positions that affect specific channel features
The amino acid substitutions, together with the different physiological properties found in the six new Kcv channels, guided site-directed mutations, either singly or in combinations

Summary

EXPERIMENTAL PROCEDURES

Mutagenesis and Expression of the kcv Genes in Oocytes—Kcv cDNA was cloned into pSGEM vector Electrophysiology—A two-electrode voltage clamp (Gene clamp 500; Axon Instruments) was used to record Kϩ currents from oocytes. Ion Permeability—Permeability ratios (PB/PA) were calculated using the equation ⌬Erev ϭ Erev,B Ϫ Erev,A ϭ RT/zF ln PB[B]o/PA[A]o, where Erev is the value in millivolts of the current reversal potential measured in the presence of 50 mM monovalent cation (either [A]o or [B]o) in the external solution, R is the gas constant, T is the absolute temperature, z is the ion charge, and F is Faraday’s constant (1). Analogy Modeling—Multiple sequence alignments of KCV, KirBac1.1, and various other Kϩ channels were performed with ClustalX v1.8 software. The final alignment was used to match the Kcv sequence to the three-dimensional template of KirBac1.1.

RESULTS

DISCUSSION

CONCLUSIONS