Development of a K(+)-channel probe and its use for identification of an intracellular plant membrane K+ channel.
Polyclonal antibodies were generated against a 9-amino acid, synthetic peptide corresponding to the selectivity filter in the pore region of K(+)-channel proteins. The sequence of amino acids in the ion-conducting pore region of K+ channels is the only highly conserved region of members of this protein family. The objectives of the present work were (i) to determine whether the anti-channel pore peptide antibody was immunoreactive with known K(+)-channel proteins and (ii) to demonstrate the usefulness of the antibody by employing it to identify a newly discovered K(+)-channel protein. Anti-channel pore peptide was immunoreactive with various K(+)-channel subtypes native to a number of different species. Immunoblot analysis demonstrated affinity of the antibody for the drk1, maxi-K, and KAT1 K(+)-channel proteins. Studies also suggested that the anti-channel pore peptide antibody did not immunoreact with membrane proteins other than K+ channels. The anti-channel pore peptide antibody was used to establish the identity of a 62-kDa chloroplast inner envelope polypeptide as a putative component of a K(+)-channel protein. It was concluded that an antibody generated against the conserved pore region/selectivity filter of K+ channels has broad but selective affinity for this class of proteins. This K(+)-channel probe may be a useful tool for identification of K(+)-channel proteins in native membranes.
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Plant Science Department, Cook College, Rutgers State University of New Jersey, New Brunswick 08903, USA.
Berkowitz
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GA
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D002417:Cattle; D002478:Cells, Cultured; D002736:Chloroplasts / Q000737:chemistry* / Q000276:immunology; D017124:Conserved Sequence; D006801:Humans; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000737:chemistry* / Q000276:immunology; D015335:Molecular Probes; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D010940:Plant Proteins / Q000276:immunology / Q000302:isolation & purification*; D010944:Plants / Q000276:immunology; D015221:Potassium Channels / Q000276:immunology* / Q000302:isolation & purification*
D015335:Molecular Probes; D010446:Peptide Fragments; D010940:Plant Proteins; D015221:Potassium Channels