A K+-conducting protein of the chloroplast inner envelope was characterized as a K+ channel. Studies of this transport protein in the native membrane documented its sensitivity to K+ channel blockers. Further studies of native membranes demonstrated a sensitivity of K+ conductance to divalent cations such as MgZ+, which modulate ion conduction through interaction with negative surface charges on the inner-envelope membrane. Purified chloroplast inner-envelope vesicles were fused into an artificial planar lipid bilayer to facilitate recording of single-channel K+ currents. These single-channel K+ currents had a slope conductance of 160 picosiemens. Antibodies generated against the conserved amino acid sequence that serves as a selectivity filter in the pore of K+ channels immunoreacted with a 62-kD polypeptide derived from the chloroplast inner envelope. This polypeptide was fractionated using density gradient centrifugation. Comigration of this immunoreactive polypeptide and K+ channel activity in sucrose density gradients further suggested that this polypeptide is the protein facilitating K+ conductance across the chloroplast inner envelope.Studies with intact chloroplasts (Wu and Berkowitz, 1992) indicate that K+ flux across the inner envelope occurs through a well-regulated transport pathway that is likely a K+-conducting ion channel. K' flux into or out of the chloroplast stroma is indirectly linked to H+ counterexchange (Maury et al., 1981; Wu and Berkowitz, 1992). Movement of K+ through this channel protein, therefore, has a profound effect on stromal pH and, hence, photosynthesis (Werdan et al., 1975;Maury et al., 1981; Wu and Berkowitz, 1992). Previous work from this laboratory has demonstrated that a K'-conducting protein can be successfully detergent-solubilized from preparations of purified inner-envelope membrane vesicles and functionally reconstituted into artificial liposomes (Wang et al., 1993). This putative K+ channel protein has not previously been characterized. In the work reported here, we used severa1 approaches to characterize the nature of this K+ transport protein. Results of these experiments are consistent with the presence of a K+ channel in the inner envelope that shares some structural, functional, and regulatory properties with K' channels from other membrane systems ( e g Rudy et al., 1991; Anderson et al., 1992). 955 MATERIALS AND METHODS Preparation of Chloroplast Inner-Envelope Membrane VesiclesInner-envelope vesicles were prepared from spinach (Spinacia oleracea L.) as described previously (Berkowitz and Peters, 1993). Briefly, intact chloroplasts (isolated from 5-10 kg of spinach leaves using Perco11 step gradients) were exposed to freeze/thaw cycles in hyperosmotic medium, and the thylakoids were removed before the crude membrane fraction was loaded on a discontinuous SUC step gradient. Inner-envelope vesicles were removed from the 0.8 ~/ 0 . 4 6 M Suc interface of the gradient, washed in envelope medium (0.2 M SUC, 2 mM Na2EDTA, 2 mM DTT, and 10 m~ TricineNaOH, pH 7.5), a...

Transport studies identified a K+ channel protein in preparations of purified spinach (Spinacea oleracea) thylakoid membrane. This protein was solubilized from native membranes and reconstituted into artificial proteoliposomes with maintenance of functional integrity. A 33-kD thylakoid polypeptide was identified as a putative component of this thylakoid protein. This identification was made using an antihody raised against a synthetic peptide representing a highly conserved region of K+ channel proteins. K+ channel activity co-migrated with the immunoreactive 33-kD polypeptide when solubilized thylakoid memhrane protein was fractionated on a SUC density gradient. The antibody was used to immunoprecipitate the 33-kD polypeptide. Physiological function of this thylakoid membrane protein was elucidated hy measuring photosynthetic electron transport of thylakoid preparations in the presence and absence of a K+ channel blocker. Results indicated that K+ efflux from the thylakoid lumen through this channel protein is required for the optimization of photosynthetic capacity. The effect this protein has on photosynthetic capacity is likely due to the requirement for K+ efflux from the thylakoid lumen to charge-balance light-induced proton pumping across this membrane. The chemisomotic model of energy transduction across thylakoid membranes posited by Mitchell (1961) stands as a landmark in the evolution of our understanding of cell bioenergetics. This model was transformed from a thermo-dynamic abstraction into an explicit description of light-driven energy conservation by the demonstration of light-induced H+ pumping into the thylakoid lumen (Neumann and Jagendorf, 1964) and the coupling of ATP formation to the ApH across the thylakoid membrane (Jagendorf and Uribe, 1966). However, an important component of Mitchell's chemi-osmotic model is the electroneutral nature of vectorial proton movement across the thylakoid membrane. Energy conservation by the thylakoid was thought to be linked primarily to ApH, with electroneutrality maintained by coincident flux of other ions. Subsequent studies (Junge and Jackson, 1982) confirmed that the substantial ApH generated across membranes of illuminated thylakoids oc-'; fax 1-908-932-9441. curred in the absence of any significant hyperpolarization. A full characterization of the system that allows for the partitioning of the chemical and electrical components of photosynthetic proton pumping, thus facilitating electro-neutra1 fluxes across the thylakoid membrane, awaits further work. Numerous studies during the last severa1 decades have focused on delineating the specific ions that are involved in this electroneutral flux (Dilley and Vernon, 1965; Witt, 1971; Hind et al., 1974; Barber, 1976; Chow et al., 1976; Krause, 1977; Portis, 1981; Vambutus and Schechter, 1983; Vambutus et al., 1984). The results of these studies indicate that Mg2+ and K+ flux out of the lumen, and C1-movement into the thylakoid from the stroma occur upon illumination and, therefore, can be envisaged as potent...

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...