The b subunit of the high-conductance calcium-activated potassium channel contributes to the high-affinity receptor for charybdotoxin (transient expressionytoxin bindingyWestern blotsycrosslinking)

Transient expression of either a or a1b subunits of the high-conductance Ca21-activated K1 (maxi-K) channel has been achieved in COS-1 cells. Expression has been studied using charybdotoxin (ChTX), a peptidyl inhibitor that binds in the pore on the a subunit. Although some properties of monoiodotyrosine-ChTX (125I-ChTX) binding to membranes derived from each type of transfected cells appear to be identical, other parameters of the binding reaction are markedly different. Under low ionic strength conditions, the affinity constant for 125I-ChTX measured under equilibrium binding conditions is increased ca. 50-fold in the presence of the b subunit. The rate constant for 125I-ChTX association is enhanced ca. 5-fold, whereas the dissociation rate constant is decreased more than 7-fold when the b subunit is present. These data indicate that functional coassembly of maxi-K channel subunits can be obtained in a transient expression system, and that the b subunit has profound effects on 125I-ChTX binding. We postulate that certain negatively charged residues in the large extracellular loop ofb attract the positively charged 125I-ChTX to its binding site on a through electrostatic interactions, and account for effects observed on ligand association kinetics. Moreover, another residue(s) in the loop of b must contribute to stabilization of the toxinbound state, either by a direct interaction with toxin, or through an allosteric effect on the a subunit. Certain regions in the extracellular loop of the b subunit may be in close proximity to the pore of the channel, and could play an important role in maxi-K channel function. High-conductance Ca21-activated K1 (maxi-K) channels display characteristic biophysical and pharmacological properties that distinguish them from other ion channels. Maxi-K channels are activated by both membrane depolarization and intracellular Ca21, and exhibit a large potassium conductance (200–300 pS), as well as high selectivity for this cation. These channels are sensitive to inhibition by external application of a number of peptidyl toxins, such as charybdotoxin (ChTX) and iberiotoxin (1). Maxi-K channels are also modulated by a series of small molecule natural products; indole diterpene alkaloids such as paxilline represent the most potent and selective nonpeptidyl blockers of this channel discovered to date (2); glycosylated triterpene channel agonists, like dehydrosoyasaponin I, bind to the cytoplasmic face of the protein and enhance its activity (3). The maxi-K channel has been purified to homogeneity from tracheal (4) and aortic smooth muscle (5) and shown to be composed of two structurally distinct subunits, a and b. Although expression of the a subunit of the maxi-K channel in Xenopus oocytes yields channels that are activated by Ca21 and voltage (6), coexpression of b with the a subunit has profound effects on the biophysical and pharmacological properties of the channel. In the presence of the b subunit, there is a shift in the midpoint of channel activation of 80–100 mV in the hyperpolarized direction (7–9). This shift is equivalent to that produced by a 10-fold increase in Ca21 concentration. Thus, the b subunit may change the overall conformation of the channel in such a way that the transitions to the open state are more favored. In addition, the agonist activity of the glycosylated triterpenes requires the presence of the b subunit (8). Heterologous expression of the a subunit yields channels that are sensitive to ChTX. This is expected since ChTX binds in the pore formed by four a subunits to block the ion conduction pathway. However, possible contributions of the b subunit to the ChTX receptor have not been explored. In this study, the characteristics of the interaction of monoiodotyrosine-ChTX (125I-ChTX) with membranes derived from COS-1 cells transiently transfected with either a or a and b subunits of the maxi-K channel have been determined. The results of these studies indicate that the b subunit causes a significant enhancement in toxin affinity of ca. 50-fold through an effect on both association and dissociation kinetics. Thus, it appears that certain regions in the large extracellular loop of b are in close proximity to the toxin binding site and may even participate in it and in other aspects of channel function. MATERIALS AND METHODS Materials. Restriction enzymes and pCI-neo vector were from Promega. COS-1 cells (culture CRL1650) were obtained from American Type Culture Collection. All tissue culture media and the LipofectAMINE reagent were from GIBCO. Fetal bovine serum was purchased from HyClone. Recombinant N-glycanase was from Genzyme. Digitonin special grade (water soluble) was obtained from Biosynth Technologies (Skokie, IL). GFyC glass fiber filters were purchased from Whatman. Affinity-purified alkaline phosphatase-conjugated goat anti-rabbit IgG, nitroblue tetrazolium (NBT), 5-bromo4-chloro-3-indoyl phosphate p-toluidine salt (BCIP), Tween 20, Triton X-100, and paxilline were obtained from Sigma. Disuccinimidyl suberate (DSS) was from Pierce. ChTX was obtained from Peninsula Laboratories and radioiodinated as described (10). 125I-Na was obtained from DuPontyNEN. 49,6-Diamidino-2-phenylindole was from Molecular Probes. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA 0027-8424y97y942853-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: ChTX, charybdotoxin; 125I-ChTX, monoiodotyrosineChTX; maxi-K channel, high-conductance Ca21-activated K1 channel; DSS, disuccinimidyl suberate. ‡To whom reprint requests should be addressed.