DEVELOPMENT OF A K-CHANNEL PROBE AND ITS USE FOR IDENTIFICATION OF ANINTRACELLULAR 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-conductin g pore region of K+ channels is the only highly conserved region of me mbers of this protein family. The objectives of the present work were (i) to determine whether the anti-channel pore peptide antibody was im munoreactive with known K+-channel proteins and (ii) to demonstrate th e usefulness of the antibody by employing it to identify a newly disco vered K+-channel protein. Anti-channel pore peptide was immunoreactive with various K+-channel subtypes native to a number of different spec ies, Immunoblot analysis demonstrated affinity of the antibody for the drk1, maxi-K, and KAT1 K+-channel proteins. Studies also suggested th at the anti-channel pore peptide antibody did not immunoreact with mem brane proteins other than K+ channels. The anti-channel pore peptide a ntibody was used to establish the identity of a 62-kDa chloroplast inn er envelope polypeptide as a putative component of a K+-channel protei n. It was concluded that an antibody generated against the conserved p are region/selectivity filter of K+ channels has broad but selective a ffinity for this class of proteins. This K+-channel probe may be a use ful tool for identification of K+-channel proteins in native membranes .