A neuronal ␤ subunit (KCNMB4) makes the large conductance, voltage- and Ca2؉-activated K؉ channel resistant to charybdotoxin and iberiotoxin Pratap Meera*†, Martin Wallner*†, and Ligia Toro*‡§ Departments of *Anesthesiology and ‡Molecular and Medical Pharmacology and the Brain Research Institute, University of California, Los Angeles, CA 90095-7115 Communicated by Ramon Latorre, Center for Scientific Studies of Santiago, Valdivia, Chile, March 15, 2000 (received for review February 4, 2000) Large conductance voltage and Ca2؉-activated K؉ (MaxiK) chan- magnocellular neurons a CTx-sensitive isoform is localized on nels couple intracellular Ca2؉ with cellular excitability. They are the cell body (22), whereas a CTx-insensitive isoform is present composed of a pore-forming ␣ subunit and modulatory ␤ subunits. at the nerve endings (23). In addition, MaxiK currents in other The pore blockers charybdotoxin (CTx) and iberiotoxin (IbTx), at neuronal preparations are only partially blocked by CTx and nanomolar concentrations, have been invaluable in unraveling IbTx (15, 24). MaxiK channel physiological role in vertebrates. However in mam- Herein, we report that coexpression of a neuronal MaxiK malian brain, CTx-insensitive MaxiK channels have been described channel ␤ subunit (␤4) leads to a MaxiK channel phenotype that [Reinhart, P. H., Chung, S. & Levitan, I. B. (1989) Neuron 2, 1031– displays a low apparent IbTx and CTx sensitivity due to dramat- 1041], but their molecular basis is unknown. Here we report a ically decreased toxin association rates, by Ϸ250–1,000 fold. human MaxiK channel ␤-subunit (␤4), highly expressed in brain, Exchange of the extracellular loop between the smooth muscle which renders the MaxiK channel ␣-subunit resistant to nanomolar ␤1 and neuronal ␤4 subunits shows that this domain is respon- concentrations of CTx and IbTx. The resistance of MaxiK channel to sible for toxin resistance. We propose that the assembly of the toxin block, a phenotype conferred by the ␤4 extracellular loop, neuronal MaxiK channel ␤4 subunit with its pore forming ␣ results from a dramatic (Ϸ1,000 fold) slowdown of the toxin subunit in the brain forms the molecular basis for toxin- association. However once bound, the toxin block is apparently insensitive MaxiK channels found in neurons. irreversible. Thus, unusually high toxin concentrations and long exposure times are necessary to determine the role of ‘‘CTx͞IbTx- Experimental Procedures insensitive’’ MaxiK channels formed by ␣ ؉ ␤4 subunits. Cloning and Expression. The MaxiK ␤3 and ␤4 subunits were identified in the expressed sequence tag (EST) database. ␤3 axiK (large conductance, voltage- and Ca2ϩ-activated Kϩ) (accession no. AF160968) and ␤4 (accession no. AF160967) Mchannels are composed of a pore-forming ␣ subunit (1) cDNA clones were isolated from an arrayed, normalized human and modulatory transmembrane ␤ subunits (2–4). Association cDNA library (␤3, L12-D12; ␤4, F23F16; HUCL-Array library, with tissue-specific modulatory ␤ subunits may account for much Stratagene). The ␤3 clone contains several in-frame upstream of the phenotypic MaxiK channel variability observed in native stop codons before the start methionine. The sequence upstream tissues (5). ␤ subunits increase the apparent Ca2ϩ͞voltage of the Kozak consensus sequence (25) of ␤4 clone F23F16 is sensitivity of the ␣ subunit (3, 6, 7), modify channel kinetics unusually GC-rich (82.5% G ϩ C in 337 bases), a feature (8–10), and alter its pharmacological properties (3, 11). MaxiK frequently found in 5Ј-untranslated regions, and lacks an up- channel ␤1 and ␤2 subunits can alter the charybdotoxin (CTx) stream in-frame stop codon. For functional expression and in and iberiotoxin (IbTx) interaction in electrophysiological (9–11) vitro translation experiments, the untranslated (UT) regions and biochemical (12) studies; however, none of the reported ␤ were removed and replaced with 182 bp 5Ј-UT region from the subunits can account for the toxin-insensitive MaxiK channels Shaker potassium channel. The ␤45Ј-GC-rich region was con- found in brain (13). firmed by genomic sequencing (P1 clone, Genome Systems, St. MaxiK channels are important regulators of neuronal func- Louis). The genomic sequence is identical with the ␤4 cDNA tion. In presynaptic terminals, MaxiK channels are found colo- sequence from position 1 to 673 (ends at CCAAG). After calized with voltage-dependent Ca2ϩ channels and, therefore, position 673 of the cDNA sequence, the genomic clone contains are thought to play a critical role in the regulation of neuro- an intron. transmitter release (14–17). However, because of the lack of The ␤3⌬N38-ball construct contained the N-terminal 19-aa IbTx effects in striatal brain slices (18, 19), the general involve- ‘‘ball’’ from the ␤2 subunit (9), a 7-aa linker (AMGRHEG) ment of MaxiK channels in neurotransmitter release remains followed by ␤3 (from amino acid 39). Chimeric ‘‘␤1–␤4 loop uncertain. IbTx is considered to be a specific MaxiK channel blocker, 2ϩ ϩ whereas CTx also blocks other potassium channels (20). Because Abbreviations: MaxiK channel, large conductance, voltage- and Ca -activated K chan- nel; CTx, charybdotoxin; IbTx, iberiotoxin. of its specificity, IbTx is used as a pharmacological agent to Data deposition: The sequences reported in the paper have been deposited in the GenBank dissect the role of MaxiK channels in physiological processes, database [accession nos. AF160968 (KCNMB3) and AF160967 (KCNMB4)]. and generally it is used at a concentration of 100 nM. In contrast †P.M. and M.W. contributed equally to this work. to smooth and skeletal muscles, where MaxiK channels are §To whom reprint requests should be addressed. E-mail: [email protected]. normally blocked by 100 nM IbTx or CTx, in neurons both The publication costs of this article were defrayed in part by page charge payment. This CTx-sensitive and -insensitive MaxiK channels are found. CTx- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. insensitive MaxiK channels were first described in rat brain §1734 solely to indicate this fact. membrane preparations (13) and later in embryonic telence- Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.100118597. phalic neuroepithelium (21). Interestingly, in rat supraoptic Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.100118597 5562–5567 ͉ PNAS ͉ May 9, 2000 ͉ vol. 97 ͉ no. 10 Downloaded by guest on September 29, 2021 exchange’’ constructs ␤1L␤4 and ␤4L␤1 were made using the ball) yielded inactivating currents suggesting that it interacts with overlap extension method (26). The predicted protein sequence the ␣ subunit (not shown). can be deduced from the sequence alignments (Fig. 1) by The similarity of the ␤4 and ␤3 proteins with the other swapping the sequence between transmembrane regions TM1 members of the MaxiK channel ␤ subunit family suggests a and TM2 between ␤1 and ␤4. DNA was sequenced on both common structure with two membrane spanning regions (TM1 strands. Northern and Dot blots (CLONTECH) were hybridized and TM2) separated by a large extracellular loop (Fig. 1a and at high stringency (9). Internal standards were ␤1, ␤2, and ␤4 Fig. 3b, Inset). The ␤4 protein has a consensus sequence for cRNA transcripts (in 200 ng of human placenta total RNA as protein kinase A (PKA) phosphorylation at the C terminus; carrier) spotted on Nytran Plus (Schleicher & Schuell) mem- whereas ␤1, ␤2, and ␤3 carry PKA sites at the N terminus. The branes. The internal standards were processed together with ␤4 and ␤3 extracellular loops contain two consensus sequences the dot blot membranes, and signals were quantified with a for N-linked glycosylation, one of these sites is conserved in all phosphorimager. four human ␤ subunit homologs, and the other is unique (Fig. 1a). Remarkable is the conservation of four cysteine residues Chromosomal Localization. The bacteriophage P1 clone carrying among the ␤ subunit family (12). A dendrogram illustrating the the human genomic ␤4 gene hybridized to the long arm of sequence similarities among ␤-subunit proteins shows that ␤4is chromosome 12 using fluorescent in situ hybridization (Genome closer to the ␤3 subunit than to the ␤1 and ␤2 subunits (Fig. 1b). Systems). This localization was confirmed by cohybridization of To confirm the proposed structural features of the ␤4 and ␤3 the digoxigenin-labeled ␤4 genomic clone with a biotin-labeled subunits, we performed in vitro translation in the presence of probe specific for the centromere of chromosome 12. Hybrid- microsomes and deglycosylation with N-glycosidase F. Electro- ization was visualized with a fluoresceinated antidigoxigenin phoretic size separation of in vitro translated ␤-subunit proteins antibody (␤4-probe) and Texas red avidin for the chromosome leads to bands of the expected molecular mass (Fig. 1c). Treat- 12 centromere-specific probe. ment with N-glycosidase (Fig. 1c, ϩ) reduces the apparent molecular weight by Ϸ8 kDa (␤1, ␤3, and ␤4) and Ϸ12 kDa (␤2), Electrophysiology. Xenopus laevis oocytes were injected with 2 ng consistent with the number of consensus sequences for N-linked of human MaxiK ␣ subunit (hslo; GenBank accession no. glycosylation. Therefore, it is likely that all N-linked glycosyla- U11058) and 4 ng of ␤ subunit cRNAs, which corresponded to tion sites in these MaxiK ␤ subunits are available for N- a Ϸ6 fold molar excess of ␤ cRNA. Based on our previous studies glycosylation in the native protein. with other ␤ subunits, it is likely that ␤3 and ␤4 subunits are at saturating concentrations. HEK293T cells were transfected with The MaxiK Channel ␤4 Subunit mRNA Is Abundant in Brain. Dot blot ␤4 in pcDNA3. Pipette and bath solutions (mM): 110 Kϩ- and Northern blot analysis show strong ␤4 signals in all brain methanesulfonate, 5 KCl, 10 Hepes, 5 N-(2-hydroxyethyl)ethyl- regions (Fig. 2a, lanes A and B, G1) with lower signals in many enediamine triacetic acid, pH 7.0; patch pipettes had resistances other tissues. Dot blot quantification (Fig. 2b) using an internal of 1–2 M⍀. Free Ca2ϩ concentrations were calculated with standard with known amounts of ␤-subunit transcripts (Fig.