Development of a K(+)-channel probe and its use for identification of an intracellular plant membrane K+ channel.

Abstract

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 drkl, 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.K+ channels are a diverse and ubiquitous family of proteins. Over the past few years, homology screening of cDNA libraries and expression cloning (in Xenopus oocytes or yeast null mutants) have led to identification of nucleotide sequences encoding numerous newly discovered K+ channels. However, virtually everything we know about the structure of these proteins is deduced from analysis of the corresponding nucleotide sequences. Few molecular analyses have been undertaken on K+-channel proteins expressed in native membranes. This is due to the lack of useful probes with strong and specific affinity for a broad range of K+-channel proteins. Radiolabeled neurotoxins such as charybdotoxin and dendrotoxin have aided the study of a few K+-channel proteins (1, 2). However, these toxins do not bind to all K+-channel types and have been found to have no affinity for plant K+ channels (3). Here, we describe an alternative approach to identification of newly discovered members of this protein family. This research focuses on the development and testing of a "generic" K+-channel antibody. The strategy we followed was to raise polyclonal antibodies to a peptide corresponding to a highly conserved region of K+ channels.The K+-conducting portion of native K+ channels is thought to be a multimer of four identical or similar polypeptides (4). Recent reviews group known K+-channel polypeptides into four subfamilies (4,5). Deduced sequences of K+ channels belonging to the four major groupings share some structural homology. There is, however, virtually no region of the The publication costs of this article were defrayed in part by page charge paymen...

Myelination and Regional Domain Differentiation of the Axon

“…It has been proposed that the significance of localizing potassium channels in this domain is concerned with repolarization of the action potential, as well as counteracting instability of excitability, especially in the transition zone between myelinated and unmyelinated portions of distal motor nerve fibers (Chiu et al 1999; Rasband et al 1998; Zhou et al 1998b). Specifically, two rectifying Shaker -like potassium channels, Kv1.1 and Kv1.2, occupy a majority of the domain, along with the CAMs, Caspr2, and TAG-1 (Arroyo et al 1999; Mi and Berkowitz 1995; Rasband et al 1998; Wang et al 1993). Caspr2 is expressed strictly in the axon and is functionally related to Caspr, except that it contains a PDZ binding motif in its intracellular domain that Caspr lacks (Poliak et al 1999).…”

Functional expression and characterization of a plant K+ channel gene in a plant cell model

“…In the past decade, studies on ion transport in plants have been advanced by molecular studies of ion pumps and channels Czempinski et al, 1997;Mi and Berkowitz, 1995;Mueller-Roeber et al, 1995;Schachtman and Schroeder, 1994;Sentenac et al, 1992;. Sussman, 1991).…”