Molecular basis of fast inactivation in voltage and Ca 2 1-activated K 1 channels : A transmembrane b-subunit homolog

Voltage-dependent and calcium-sensitive K1 (MaxiK) channels are key regulators of neuronal excitability, secretion, and vascular tone because of their ability to sense transmembrane voltage and intracellular Ca21. In most tissues, their stimulation results in a noninactivating hyperpolarizing K1 current that reduces excitability. In addition to noninactivating MaxiK currents, an inactivating MaxiK channel phenotype is found in cells like chromaffin cells and hippocampal neurons. The molecular determinants underlying inactivating MaxiK channels remain unknown. Herein, we report a transmembrane b subunit (b2) that yields inactivating MaxiK currents on coexpression with the pore-forming a subunit of MaxiK channels. Intracellular application of trypsin as well as deletion of 19 N-terminal amino acids of the b2 subunit abolished inactivation of the a subunit. Conversely, fusion of these N-terminal amino acids to the noninactivating smooth muscle b1 subunit leads to an inactivating phenotype of MaxiK channels. Furthermore, addition of a synthetic N-terminal peptide of the b2 subunit causes inactivation of the MaxiK channel a subunit by occluding its K1-conducting pore resembling the inactivation caused by the ‘‘ball’’ peptide in voltage-dependent K1 channels. Thus, the inactivating phenotype of MaxiK channels in native tissues can result from the association with different b subunits. Ca21-activated K1 channels, also known as BK, MaxiK, or slo channels, are key modulators of cellular excitability. They are characterized by their large single-channel conductance, intrinsic voltage dependence, Ca21 modulation, and blockade by charybdotoxin (CTX) and iberiotoxin (1–5). In most tissues, MaxiK channels produce noninactivating currents when activated by depolarization andyor an increase in intracellular Ca21. However, in chromaffin cells of the adrenal gland (6) and hippocampal neurons (7), inactivating MaxiK currents are also observed that otherwise resemble their noninactivating counterparts in their biophysical and pharmacological properties. The mechanism of inactivation in MaxiK channels has been investigated in detail in rat chromaffin cells, which express both inactivating and noninactivating MaxiK channels (8). Inactivation is removed by trypsin application to the cytosolic face of the membrane, suggesting the presence of an associated cytosolic inactivating particle (6, 7). A model developed to explain the biophysical and pharmacological properties of inactivating channels in chromaffin cells suggests that these channels are formed by a tetrameric assembly of inactivating and noninactivating subunits. Interestingly, inactivating channels in chromaffin cells are less sensitive to CTX, and heteromeric channels consisting of inactivating and noninactivating isoforms seem to have intermediate toxin sensitivities (9). Identification of a MaxiK channel variant or a subunit capable of producing fast inactivating MaxiK channel currents has so far been elusive (10). Here, we report a human b subunit (b2) that yields fast inactivating currents when coexpressed with the human MaxiK channel a subunit (hslo), similar to those found in native cells. Deletion and fusion constructs demonstrate that the inactivating particle is located at the intracellular N terminus of this transmembrane b2 subunit. An N-terminal synthetic peptide of this b subunit induces inactivation of MaxiK channels, which resembles N-type inactivation of voltage-dependent K1 channels by ‘‘ball’’ peptides. Thus, N-type inactivation is conferred to MaxiK channels by a ‘‘ball’’ peptide attached to the intracellular N terminus of their associated b subunits. MATERIALS AND METHODS Molecular Biology. Expressed sequence tag (EST) databases were searched with the BLAST algorithm with the b1 subunit (GenBank accession no. U25138) as a query sequence. Clone oe72h09 was obtained from the American Type Culture Collection and was sequenced on both strands by using fluorescent sequencing (GenBank accession no. AF099137, b2 subunit). The sequence alignment was performed with the GCG program GAP by using default values. Linearized clones were used for in vitro transcription (mMESSAGEmMACHINE, Ambion, Austin, TX). cRNA (0.5 mg) was in vitro translated in 25 ml of reticulocyte lysate in the presence of microsomal membranes (Promega) and [35S]methionine. After a 1-hr incubation at 30°C, the microsomal membranes were collected by centrifugation and an aliquot was treated with N-glycosidase F (NEB, Beverly, MA). Proteins were separated by SDSyPAGE and visualized with a PhosphorImager (Molecular Dynamics). Dot and Northern blots were hybridized according to the manufacturer’s (CLONTECH) instructions, except that Cot-1 DNA was omitted from the hybridization mix. Briefly, at least 20-min prehybridization was followed by overnight hybridization at 65°C with 32P-labeled b2 DNA fragment. The membranes were washed at 55°C with 0.1 3 SSCy0.5% SDS. Internal standards were prepared by spotting dilutions of b1 and b2 cRNA (in 100 ng human placenta total RNA as a carrier) on nylon membranes (Nytran plus, Schleicher & Schuell), following the protocol used for preparation of the commercial dot-blot membranes. After hybridization and washing, the membranes were analyzed using PhosphorImaging (Molecular Dynamics). Clone b2-IR (inactivation removed) was made by deleting 19 N-terminal amino acids from the b2 subunit and introducThe 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. PNAS is available online at www.pnas. org. This paper was submitted directly (Track II) to the Proceedings office. Abbreviations: MaxiK, large conductance, voltage and Ca21-activated K1 channel; hslo, human MaxiK channel a subunit; DHS-I, dehydrosoyasaponin I; CTX, charybdotoxin; TEA, tetraethylammonium; b2, transmembrane b subunit; EST, expressed sequence tag; IR, inactivation removed. Data deposition: The sequence reported in this paper has been deposited in GenBank database (accession no. AF099137). *These authors contributed equally to this work. §To whom reprint requests should be addressed at: Department of Anesthesiology, University of California, BH-612 CHS, Box 951778, Los Angeles, CA 90095-1778. e-mail: ltoro@ucla.edu.