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THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 275, No. 9, Issue of March 3, pp. 6453–6461, 2000 Printed in U.S.A.

Cloning and Functional Characterization of Novel Large Conductance Calcium-activated Potassium Channel ! Subunits, hKCNMB3 and hKCNMB4* (Received for publication, November 4, 1999, and in revised form, December 18, 1999)

Robert Brenner‡, Tim J. Jegla§, Alan Wickenden§, Yi Liu§, and Richard W. Aldrich‡¶ From the ‡Howard Hughes Medical Institute, Molecular and Cellular Physiology, Stanford School of Medicine, Stanford, California 94305 and §Icagen, Durham, North Carolina 27703

The large conductance calcium-activated potassium channel (BK)1 is a unique member of the six transmembrane domain potassium channel family that is activated by voltage and calcium. BK channels are composed of a pore-forming ! subunit * This work was supported by Grant NS23294 from the National Institutes of Health and by Grant MH48108 from the National Institute of Mental Health Silvio Conte Center for Neuroscience Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF209747 (for H"2), AF214561 (for H"3), and AF207992 for (H"4). ¶ Investigator with the Howard Hughes Medical Institute. To whom correspondence should be addressed: Dept. of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305. Tel.: 650-723-6531; Fax: 650725-4463; E-mail: [email protected]. 1 The abbreviations used are: BK, large conductance calcium-activated potassium channel; slo, slowpoke; M"1, mouse "1 subunit; H"1 through H"4, human "1 through human "4 subunits; HF1, myc epitope-tagged human slo; Hslo, human slo; EST, expressed sequence tag; PCR, polymerase chain reaction; H"2!, H"2 without the inactivation domain; N-type, ball and chain inactivation mechanism; V1⁄2, halfactivation voltage; KCNMB1 through KCNMB4, "1 through "4 subunit genes; kb, kilobase pair. This paper is available on line at http://www.jbc.org

(1, 2) and, in some tissues, are tightly associated with an accessory " subunit (3, 4). BK channels have diverse physiological properties with tissue-specific distribution. In neurons, BK channels are functionally colocalized with calcium channels (5, 6), shape action potential wave forms (7, 8), and modulate neurotransmitter release (9, 10). In smooth muscle, BK channels regulate constriction in arteries (11), uterine contraction (12), and filtration rate in the kidney (13). Unlike other potassium channel families, BK channels can as yet only be attributed to a single gene, slowpoke (slo), that encodes the poreforming ! subunit of the channel. In light of the broad tissue localization and diverse functional properties, it is not surprising that a number of mechanisms have been identified that alter slo channel properties. These include alternative splicing of the slo RNA (14 –18), heteromeric assembly with other subunits (slak) (19), and modification by phosphorylation/dephosphorylation (20 –22) and oxidation/reduction (23). In addition, accessory " subunits are a means of generating BK channel diversity. Coexpression of the "1 subunit in Xenopus oocytes increases the apparent calcium sensitivity, slows activation kinetics, and increases charybdotoxin binding affinity (24 –27). "1 subunit mRNA is enriched in smooth muscle (28) and can account for the apparent increased calcium sensitivity of BK channels in that tissue relative to skeletal muscle (29, 30) where the "1 gene shows little expression. In the chick cochlea, a spatial gradient of " subunit expression may contribute to the slo channel diversity that underlies electrical tuning (31). Evidence indicates that additional " subunits may associate with slo channels in different tissues. In brain tissue, biochemical purification of slo channels indicates that a novel 25-kDa subunit is tightly associated with slo ! subunits (32). Recently, a second " subunit homologue, "2, has been cloned that causes inactivation of slo channels in adrenal chromaffin cells (33, 34). However, the higher molecular weight of this "2 subunit indicates it is distinct from the brain " subunit. In an effort to understand the function of " subunits, we have cloned two novel " subunit family members, which we call H"3 (KCNMB3) and H"4 (KCNMB4). We demonstrate that these subunits interact with and modulate slo channels. In addition, we compare and contrast the four members of the " subunit family ("1 through "4) for their effects on slo channel properties. We find that M"1 and H"2 have very similar effects on slo currents differing mainly in the ability of H"2 to cause inactivation. In contrast, H"3 and H"4 have distinct and novel effects on slo currents. Our results indicate that the " subunit gene family contributes a high degree of diversity to the functional properties of slo channels. EXPERIMENTAL PROCEDURES

cDNA Cloning and Construction of " Subunits—In order to identify novel " subunit genes, a sequence similarity search was conducted

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We present the cloning and characterization of two novel calcium-activated potassium channel ! subunits, hKCNMB3 and hKCNMB4, that are enriched in the testis and brain, respectively. We compare and contrast the steady state and kinetic properties of these ! subunits with the previously cloned mouse !1 (mKCNMB1) and the human !2 subunit (hKCNMB2). Once inactivation is removed, we find that hKCNMB2 has properties similar to mKCNMB1. hKCNMB2 slows Hslo1 channel gating and shifts the current-voltage relationship to more negative potentials. hKCNMB3 and hKCNMB4 have distinct effects on slo currents not observed with mKCNMB1 and hKCNMB2. Although we found that hKCNMB3 does interact with Hslo channels, its effects on Hslo1 channel properties were slight, increasing Hslo1 activation rates. In contrast, hKCNMB4 slows Hslo1 gating kinetics, and modulates the apparent calcium sensitivity of Hslo1. We found that the different effects of the ! subunits on some Hslo1 channel properties are calcium-dependent. mKCNMB1 and hKCNMB2 slow activation at 1 "M but not at 10 "M free calcium concentrations. hKCNMB4 decreases Hslo1 channel openings at low calcium concentrations but increases channel openings at high calcium concentrations. These results suggest that ! subunits in diverse tissue types fine-tune slo channel properties to the needs of a particular cell.

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using the NCBI Blast program. The M"1 (mouse KCNMB1) peptide sequence was used as a query sequence against the human EST DNA data base translated in all reading frames (tblastn program). Three novel classes of EST sequences representing different " subunit genes were identified. The EST clone AA904191 (IMAGE clone number 1417217) encodes a H"2 cDNA containing a complete open reading frame and is the clone we used for oocyte expression of H"2. This is the same clone that has been reported previously (33, 34). To ensure that this clone did not contain any cloning artifacts, we used clone AA904191 as a probe to hybridize to cDNAs in a human ovary cDNA library. One million recombinants were screened, and eight positives were identified on duplicate lifts. Five clones were purified, two of which contained complete open reading frames and represent a overlapping cDNA size of 2.55 kb. The cDNA was sequenced on both strands using custom primers (submitted under GenBankTM accession number AF209747), and no differences were observed in amino acid sequence between the EST clone and the human ovary clones. To make an H"2 expression clone lacking the inactivation ball, the H"2 cDNA was amplified with primers encompassing amino acids 31 to the termination codon. The upstream primer included a Kozak’s consensus translation initiation site and a glycine to precede the H"2 sequence. The PCR product was cloned into a oocyte expression vector and sequenced in its entirety. This expression construct is referred to H"2! throughout this paper. H"3 was identified from M"1 alignment to a large number of partially spliced clones and was the most abundant " subunit homologue in the EST data base. The longest and most complete H"3 EST clone that we identified was AA761761 (IMAGE clone number 1288005). Clone AA761761 encodes amino acids 16 –185 of the H"3 peptide. This clone was used to screen 106 recombinants in a human fetal brain cDNA library. Three independent positives were purified and sequenced of which one 1.8-kb clone contained a complete H"3 open reading frame (submitted under GenBankTM accession number AF214561). To confirm the H"3 5! sequence, additional H"3 cDNAs were amplified using vector (5! primer) and internal H"3 primers (3! primer) from testis and hippocampus cDNA libraries. The position of the translation initiation

site was confirmed by an in-frame stop codon 27 nucleotides upstream of the initiation methionine. Although the nucleotides surrounding the initiating methionine do not conform to an ideal translation initiation site (35), the next downstream methionine occurs well beyond regions of conservation and into the first transmembrane. To test H"3 interactions with Hslo1, the inactivating 33 amino-terminal residues of H"2 was appended to the H"3 cDNA amino terminus using overlap extension PCR (36, 37). We found that this construct produced very slow to almost undetectable inactivation of Hslo1 currents (data not shown). Sequence alignments of H"2 to H"3 indicate that the inactivation ball needs to be positioned 24 residues carboxyl-terminal to the initiating methionine to reside in a homologous position in H"3 (see Fig. 1A). A second construct, inactivation ball-"-(1–24)-H"3, that deletes the first 24 amino acids of H"3 and appends the 33-residue inactivation ball of H"2 was constructed and used for experiments described under “Results.” H"4 was identified by searching the EST data base with the H"1 sequence. Multiple ESTs were identified, but none of the clones appeared to be 5! complete. Clone AA418392 was sequenced and found to encode amino acids 142–210 of H"4. The 5! end of the coding sequence was cloned from human hippocampus using 5!-rapid amplification of cDNA ends PCR. Three independent clones were sequenced to confirm that no PCR mutations had been introduced. The full-length H"4 sequence has been deposited under GenBankTM accession number AF207992. The 5!-untranslated sequence of these clones contains a repetitive GC-rich region and lacks any initiation or stop codons. This sequence was confirmed and extended by alignment to genomic H"4 sequence found in the High Throughput Genomic Sequence (HTGS) data base (GenBankTM accession number AC011612). The 5! sequence is 83% GC in the 275 nucleotides of sequence and lacks any in-frame start or stop codons. The first in-frame start codon conforms to the Kozak consensus translation initiation site (35) and is the one we designated as the initiating methionine. No other in-frame start codon is observed until amino acid 131 of the H"4 sequence. The entire coding region was then amplified in a single fragment from a human hippocampus cDNA library using primers that overlapped the initiator


FIG. 1. Protein sequence comparisons of the BK ! gene family. A, sequence comparison of the H"1 (GenBankTM accession number U38907) to H"2, H"3, and H"4. Sequences were aligned using the MacVector Clustal alignment program (Oxford Molecular Group, Campbell, CA). Aligned residues are boxed in dark gray (and bold) for two or more identical residues and light gray for similar (conserved substitutions) amino acids. Putative transmembrane domains are shown boxed. White on black cysteine residues denote the four cysteines that are proposed to form disulfide bridges and are required for high affinity charybdotoxin binding (43) in the "1 subunit. The lysine residue in "1 subunits that cross-links with charybdotoxin (45) is circled and labeled above with “ChTX”. An N-linked glycosylation site that is conserved throughout the " gene family is labeled with #. Asterisks label "1 residues required for high affinity binding of charybdotoxin to Hslo1 channels (43). B, table of percent identity (value above) and percent conservation (similar plus identical residues, value below) between different human " subunits.

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methionine and stop codons. Clones were sequenced to confirm that no PCR mutations had been introduced. To append the H"2 inactivation ball to H"4, we used overlap extension PCR (36, 37). The aminoterminal 10 amino acids of H"4 were replaced with the amino-terminal 43 amino acids of H"2. Northern Blots—Human multiple tissue Northern blots were purchased from CLONTECH. The blots contain 2 $g/lane of poly(A)-purified mRNA normalized for "-actin expression. The hybridization and washes were performed under high stringency to reduce the possibility of cross-hybridization to other unknown or related genes. Specifically, the blots were hybridized overnight with 50% deionized formamide, 5# SSPE (SSPE: sodium chloride, sodium phosphate, EDTA), 10# Denhardt’s solution, 0.5% SDS, and 150 $g/ml sonicated herring sperm DNA at 42 °C with 32P random primer labeled DNA probe at one million counts per ml of hybridization solution. The blots were washed in 0.1# SSPE, 0.1% SDS at 70 °C and exposed for 3 days on a Molecular Dynamics PhosphorImager. For hybridization of H"2 and H"4, DNA probes were derived from PCR amplification products of the open reading frame portion of the cDNA. For H"3, the coding portion of the cDNA was PCR-amplified and cloned into the pCRBlunt cloning vector (Invitrogen). A 540-nucleotide EcoRV fragment (encoding amino acids 79 –257) was released and gel-purified for DNA labeling. Expression in Xenopus Oocytes—All experiments were performed with a human slo clone (GenBankTM accession number U11058 (38)) with an amino-terminal c-myc epitope tag (HF1), which was a kind gift from Ligia Toro (UCLA School of Medicine). This clone has been previously described (39) and is indistinguishable in properties from untagged Hslo1. This clone contains an 87-nucleotide insert at alternatively spliced site A but lacks other alternative exons (38). The Hslo1 expression construct was linearized with NotI and transcribed using T7 RNA polymerase. M"1, H"4, H"2!, IB-H"3, and IB-"-(1–24)-H"3 were cloned in the pOX oocyte expression vector and were linearized with NotI and transcribed with T3 RNA polymerase. H"2 was cloned in the pT7T3D-Pac and was linearized with SfiI and transcribed with T3 RNA polymerase. H"3 was cloned in pBluescript KS II, linearized with XhoI, and transcribed with T3 RNA polymerase. All cRNAs were transcribed using the mMessage mMachine kit (Ambion, Austin, TX). Oocytes were injected with 2 ng of Hslo1 RNA and 10 ng of " RNAs. An exception was for H"2 and IB-"-(1–24)-H"3 expression, which necessitated injection of 2 ng per oocyte instead. Although this resulted in a fraction of slo channels not being associated with inactivating " subunits, in our experiments, a large excess of H"2 and IB-"-(1–23)-H"3 caused a repression of slo current. Oocytes membranes expressing H"2 and H"4

constructs were assayed within 2–3 days of injections due to a difficulty in forming seals that increased with incubation time. All other constructs were assayed 2–5 days after injections. Electrophysiology—Recordings were made using the inside-out patch clamp configuration as has been previously described (40). Experiments were performed at 22 °C. Data were acquired at 20-$s intervals and filtered at 10 kHz using the Axopatch four-pole bessel filter. Data were analyzed without further filtering, but representative traces shown in the figures were digitally filtered at 2 kHz. Leak currents were subtracted after the test pulse by P/5 negative pulses from a holding potential of $120 mV. Patch pipettes (borosilicate glass VWR micropipettes, West Chester, PA) were coated with Sticky Wax (Kerr Corp., Romulus, MI) and fire-polished to approximately 0.8 to 1.0 megohms resistance. The external recording solution (electrode solution) was composed of 20 mM Hepes, 140 mM KMeSO3, 2 mM KCl, 2 mM MgCl2, pH 7.2. Internal solutions were composed of a pH 7.2 solution of 20 mM Hepes, 140 mM KMeSO3, 2 mM KCl, and buffered with 1 mM HEDTA and CaCl2 to the appropriate concentrations to give 1, 2, and 10 $M buffered calcium solutions (as described in Ref. 34). 50 and 200 $M calcium solutions were unbuffered. For measurements of conductance in 10, 50, and 200 $M calcium concentrations, currents were elicited with 20-ms steps to various voltages, and tail currents were measured 0.2 ms after repolarization to $80 mV. Recordings in 1 and 2 $M free calcium required 50-ms duration voltage steps for channels to reach a steady state activation. For M"1 and H"2! recordings in 50 $M calcium, a significant conductance was observed at $120 mV, hence currents were not leak subtracted, and the conductance was calculated from the peak current rather than the tails. Our recordings of uninjected oocytes typically had leak current less than 200 pA for a 100-mV step, which would not significantly affect our measurements of conductance. Recordings of the inactivation ball containing H"4 (IB-H"4) were conducted with transfected cells. (Note: all other H"4 recordings were done in oocytes with solutions described above.) HEK293 cells were cotransfected with Hslo1 with or without IB-H"4 using LipofectAMINE reagent. Membrane currents were measured from inside-out patches. The compositions of the extracellular (pipette) and intracellular (bath) solutions were as follows (mM): potassium gluconate (140), MOPS (20), EGTA (1), pH 7.4, with NaOH. Free [Ca2%] was adjusted to 50 $M in the extracellular solution and 10 $M in the intracellular solution, by addition of CaCl2. [Cl$] in all solutions was adjusted to 2 mM by addition of KCl. All recordings were made at room temperature (22–24 °C). Current records were acquired at 2–10 kHz and filtered at 5 kHz.


FIG. 2. mRNA distribution of human ! genes. Northern blots were hybridized with H"2- (top row), H"3- (middle row), and H"4 (bottom row)-specific cDNA probes as described under “Experimental Procedures.” Tissue labels apply for each column of blots. For display purposes, the extreme portions of the blots where a signal was not detected are not shown. Positions of the RNA size markers are shown on the last blot (extreme right) for each hybridization.

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RESULTS

Cloning of Novel " Subunits—In order to identify BK " homologues, the mouse " amino acid sequence was aligned to the EST data base. Two classes of novel sequences were identified. The EST clones were then used as probes to clone the complete coding sequence by conventional hybridization to cDNA libraries or reverse transcriptase-PCR from tissues. We are designating the new " genes hKCNMB3 and hKCNMB4 (human KCNMB3 and human KCMB4, respectively). This is consistent with the nomenclature committee of the Human Genome Organization designation of the H"1 subunit being named KCNMB1. However, throughout the rest of the paper we will refer to the " subunits with the common names H"2 through H"4 (human "2 through "4 for hKCNMB2 through hKCNMB4, respectively) and will call the original " clone mouse gene M"1 (mouse "1). The H"2 is the inactivating " subunit recently reported as "2 (34) and "3 (33). H"3 sequence was recently identified within a region of the chromosome that is duplicated in dup(3q) syndrome (41) but has not been functionally characterized. H"4 has not been reported previously. Sequence alignments of the " family members are shown in Fig. 1A. Sequences between family members are only moderately conserved (21– 43% identical residues, see Fig. 1B) compared with orthologous "1 genes in different species, such as human and the bovine "1 subunits that are 84% identical. The highest conservation was between the H"1 and H"2 genes, whereas the H"3 and H"4 are more distantly related to these

genes and to each other. A " subunit (not shown in Fig. 1A) has been cloned from quail (42) that has been referred to "2 (33). It has the most similarity to H"1 (47% identity) but much lower conservation than that observed between mammalian " gene orthologues. As yet, it is not clear whether the quail " gene should be recognized as a H"1 orthologue diverged between human and avian species or a novel " gene. Structurally, the " gene family is conserved in many aspects. H"1 has two putative transmembrane segments, and hydrophobicity analysis using Kyte-Doolittle plots (not shown) identified homologous hydrophobic domains (regions boxed in Fig. 1A) in the other " subunits as well. Four cysteines located in the extracellular domain that have been shown to form disulfide bridges (43) in "1 are conserved in all " subunits (white on black C residues in Fig. 1A). In addition an N-linked glycosylation site positioned at residue 80 of H"1 is conserved between the family members (#, Fig. 1A). Coexpression of the bovine "1 subunit with the slo ! subunit confers an approximately 50-fold increase in charybdotoxin binding (44) and an increased sensitivity to charybdotoxin block of BK currents (27). Lys69 on the bovine "1 subunit (shown circled in Fig. 1A) has been shown to directly cross-link with charybdotoxin (45). In addition, the Leu90, Tyr91, Thr93, and Glu94 of the "1 subunit (shown with an asterisk in Fig. 1A) are required for high affinity binding of charybdotoxin to the BK channel (43). All these residues are conserved in H"1. H"2 subunits are also conserved at residues 90 –94 and contain a


FIG. 3. Examples of Hslo1 channel currents with and without coexpression of ! subunit family members. Macroscopic currents were elicited by voltage clamp of inside/out patches in 10 $M buffered calcium solution. Voltage clamp steps were preceded by a 2-s holding voltage of $80 mV and then followed with a 20-ms test voltage. For these traces, test potentials were from $80 to 0 mV in 20-mV increments and from 0 to %100 mV in 10-mV increments. After the test potentials the membrane was repolarized to $80 mV. A–F are labeled above the current traces for the subunits expressed. F shows currents recorded from coexpression of H"2!, the H"2 subunit with a deletion of the amino-terminal inactivation ball sequence.

Novel BK " Subunits

FIG. 4. H!3 and H!4 interact with Hslo1 channels. Hslo1-" subunit interaction was assayed using the H"2 inactivation ball to detect " subunit assembly with Hslo1 ! subunits. Hslo1 channels were expressed alone (A) or with normal " subunits (B, H"2; C, M"1; E, H"3; G, H"4) or with " subunits with an appended inactivation ball sequence (D, inactivation ball-M"1; F, inactivation ball-("1–24)-H"3; H, inactivation ball-H"4). Currents were recorded from mRNA injected Xenopus oocytes (A–F) or from transfected HEK293 cells (G and H) using insideout patches in an internal solution containing 10 $M buffered calcium. Inactivation was assayed by voltage steps to %80 mV for 600 ms from a holding potential of $80 mV.

tion both occur. In subsequent experiments we used H"2 with the inactivation domain removed (H"2!) to eliminate contamination of the measurement by inactivation. At this calcium concentration, H"4 traces show the most dramatic effects on slowing of activation kinetics accompanied by a slowing of deactivation (Fig. 3E). From a cursory observation, there appears no obvious change in Hslo1 currents with coexpression of H"3 (Fig. 3D). However, a more detailed analysis (presented below) demonstrates that H"3 does interact with and modulate Hslo1 channels. H"3 and H"4 Interact with Hslo1 Channels—Expression of Hslo alone (Fig. 4A) or coexpression of the M"1 subunit (Fig. 4C) does not cause N-type inactivation of Hslo1 currents. Inactivation is observed by coexpression of H"2 (Fig. 4B), or inactivation can be conferred on M"1, by appending the H"2 inactivation ball (first 33 amino acids of H"2) onto M"1 (Fig. 4D) (33, 34). Thus we placed the H"2 inactivation ball on the other " subunits, and we used inactivation as an assay for specific interaction with Hslo1. A construct was made for H"3 that deletes the amino-terminal 24 residues and places the H"2 inactivation ball in a homologous position relative to H"2. Currents from this construct (IB-"-(1–24)-H"3, shown in Fig. 4F) show inactivation of Hslo1 currents, indicating specific interaction of H"3 with Hslo1. Inactivation by a N-type mech-


Lys107 that may be in a homologous position to the bovine "1 Lys69. However, H"1/slo and H"2/slo channels either do not affect charybdotoxin sensitivity (24) or have a reduced sensitivity to charybdotoxin, respectively, relative to slo alone (33, 34). This indicates other residues not conserved between bovine "1 and H"1 and H"2 are also important in charybdotoxin binding. H"3 and H"4 lack conserved residues at the Lys69, Tyr91, and Thr93 positions of H"1 protein and may be expected to reduce or not affect charybdotoxin binding to slo channels. Tissue Distribution of " Subunit mRNA—The "1 subunit expression is enriched in smooth muscle, particularly aorta, but shows very little expression in brain or other tissues that do not contain smooth muscle (25, 28). In order to determine where H"2, H"3, and H"4 are expressed, we hybridized specific cDNA probes for each to a battery of mRNAs on Northern blots (Fig. 2). The H"2 probe hybridized to 3 mRNA species, a strong 2.8-kb mRNA and weaker 1.6- and 6-kb mRNAs (the 6-kb band is not shown in Fig. 2). The intensity of the different bands appears to be proportional in different tissues and is likely due to different size mRNA from the same gene. Expression is highest in the ovary and relatively weaker in a large number of tissues, including heart, brain, kidney, and other tissues. Interestingly, H"2 appears to be broadly but weakly expressed throughout regions of the brain. We also saw weak expression in the adrenal gland, where H"2 is proposed to confer inactivation of BK currents (33). Hybridization with a H"3 probe showed a strong 3-kb mRNA that is restricted to testis (seen as a 3-kb band in Fig. 2). An additional, very weak, 4-kb band is observed in most nonneural tissues. In contrast, H"4 expression is broadly and strongly expressed in all neural tissues. In all brain tissue a very strong 1.9-kb band is observed, as well minor bands of 3 and 6.1 kb. Relatively moderate expression was observed in other tissues as well such as spinal cord, heart, kidney, and other tissues. It is interesting that in some tissues, such as neuronal tissues, H"2 and H"4 overlap in expression, indicating a possibility that different " subunits may coassemble with a single Hslo1 channel. Expression of Hslo1 Channels with " Subunits—The " subunit RNAs were coinjected with the Hslo1 RNA, and macroscopic currents were characterized using the inside-out patch clamp technique. Representative current traces that were evoked at voltages ranging from $80 to %100 mV in 10 $M internal calcium are shown in Fig. 3. At this calcium concentration, the Hslo1 channels (Fig. 3A) are activated relatively quickly by membrane potentials near 0 mV. Tail currents recorded at $80 mV also indicate that the channels deactivate quickly. Comparison of "-coexpressing currents shows several differences from Hslo1 currents alone. "1 subunits have been shown to shift activation of Hslo1 currents to more negative potentials and slow deactivation kinetics (24 –27, 46). This is what we observed when Hslo1 was coexpressed with M"1 and H"2! (H"2 without the inactivation ball) subunits (Fig. 3, B and F). These channels begin to activate at more negative membrane potentials, as indicated by the inward current traces. In addition tail currents are slowed dramatically and require tens of milliseconds to decay completely. The effects of H"2 on activation voltage and the slowing of deactivation kinetics are less apparent with the aminoterminal inactivation ball attached (Fig. 3C, note inactivation is not apparent within a 20-ms step but requires longer duration, as seen in Fig. 4B). It is likely that the effects of H"2 on Hslo1 channel activation and deactivation kinetics are obscured in the presence of inactivation. This is particularly apparent in the tail currents where deactivation and inactiva-

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anism in slo channels can be confirmed by removal of inactivation with trypsin digestion to the intracellular side of the channel (33, 34). We found that inactivation was also removed with trypsin digestion from the inactivation ball-("1–24)-H"3 interacting channels (data not shown). Similar experiments were used to confirm interaction of H"4 with Hslo1. Fig. 4G shows H"4 coexpression with Hslo1 does not cause inactivation of currents. However, inactivating Hslo1 currents are produced from coexpression of a inactivation ball appended H"4 (Fig. 4H), confirming interaction of H"4 with Hslo1 as well. " Subunits Alter Apparent Calcium Sensitivity—The most dramatic effect of "1 subunits on slowpoke channels is an apparent increase in calcium sensitivity (24 –27). This is readily observed as a negative shift in voltage where channels are activated at a given calcium concentration. A convenient measure of this shift is the change in voltage where one-half of maximal conductance is observed ("V1⁄2). We compared Hslo1 channel conductance alone and in the presence of the different " subunits over a broad range of calcium concentrations. Fig. 5, A—C, shows average conductance voltage curves at different calcium concentration when Hslo1 is coexpressed with the different " subunits. The absolute V1⁄2 and the change in V1⁄2 caused by coexpression of the " subunits are shown in Fig. 5, D and E, respectively. Both M"1/Hslo1 and H"2!/Hslo1 channels

are activated at more negative potentials than Hslo1 channels alone. M"1 shifted the V1⁄2 negative by 26 & 12.7, 46 & 6.9, and 47 & 3.4 mV (Fig. 5E) in 1, 10, and 50 $M free calcium concentrations, respectively. H"2! shifted the voltage range of activation even more negatively than M"1 (Fig. 5, A and E), shifting the V1⁄2 negative by 51 & 11.6, 60 & 6.9, and 57 & 3.4 mV in 1, 10, and 50 $M free calcium concentrations, respectively. On average, H"3 showed a slight negative shift of the V1⁄2 values at low calcium concentrations (Fig. 5, B and E). However, the shift was within the variance of Hslo1 currents alone and was not statistically distinguishable. H"4, in contrast to all other " subunits, decreased apparent calcium sensitivity at low calcium concentrations but increased the apparent calcium sensitivity at high calcium concentrations. This is quite evident in the conductance-voltage curves in Fig. 5, C and E, wherein H"4 coexpression shifts the curves to positive potentials in low calcium (1 $M, shifted %29 & 11.6 mV) and to negative potentials in high calcium (50 $M, shifted $29 & 3.8 mV) but has little effect in the intermediate calcium concentration of 10 $M (shifted %7.5 & 4.9 mV). Because H"4 slows activation kinetics considerably (see Fig. 6D and below) the possibility exists that the positive shift in the conductance-voltage relationship reflects a underestimate of steady state conductance before the end of the test pulse. This may be a problem at the low voltage


FIG. 5. ! subunit affects on Hslo1 conductance-voltage relations. A–C, plot of conductance versus voltage relationship for Hslo1 alone or Hslo1 coexpressed with " subunits in different calcium concentrations. Normalized conductance was measured for each test potential from current amplitude taken 200 $s after repolarization to $80 mV. Data are displayed as the mean & S.E. of multiple recordings (n is the same as the values for the V1⁄2 data in D and is given below). Solid symbols show values for Hslo1 recordings alone and are duplicated in A–C for comparison to Hslo1 % H" subunit recordings (open symbols). Recordings were made in internal solutions containing calcium concentrations of 1 $M, diamond symbols; 10 $M, square symbols; and 50 $M, circles. Solid curves show fits of data to a Boltzmann function (G ' Gmax{1/(1 % e$(V$V1/2)zF/RT)}) and then normalized to the maximum of the fit. D, plot of average V1⁄2 versus calcium concentration. V1⁄2 was calculated from Boltzmann fits of data recorded as in A. Data are displayed as the mean V1⁄2 & S.E. for the following number of data: Hslo1 alone (No ", closed box symbol), n ' 6, 12, 10, 18, and 19 for [Ca]i of 1, 2, 10, 50, and 200 $M, respectively. Hslo1 coexpressed with M"1 (upright triangle), n ' 5, 6, and 5 for [Ca]i of 1, 10, and 50 $M, respectively; Hslo1 coexpressed with H"2! (closed circles), n ' 6, 6, and 4 for [Ca]i of 1, 10, and 50 $M, respectively; Hslo1 coexpressed with H"3 (open squares), n ' 7, 7, 4, and 11 for [Ca]i of 1, 10, 50, and 200 $M, respectively; Hslo1 coexpressed with H"4 (open circles), n ' 6, 10, 10, 9, and 10 for [Ca]i of 1, 2, 10, 50, and 200 $M, respectively. E, plot of "V1⁄2 (mean V1⁄2 of Hslo1 $mean V1⁄2 of Hslo1 coexpressed with a " subunit) versus calcium concentrations. Errors were propagated by obtaining the square root of the squared sum of the errors (51). Difference in mean V1⁄2 of Hslo1 versus Hslo1 coexpressed with "s were tested using Student’s t test. All V1⁄2 values were different (p value was less than 0.05) from Hslo1 alone with the exception of Hslo1/H"3 data (p ' 0.22 to 0.40) and Hslo1/H"4 in 10 $M calcium (p ' 0.26). Hslo1/H"4 in 1 $M calcium was different with a 92% confidence (p ' 0.08).

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steps (normalized conductance ' 0.05 to 0.3), where activation time constants were from 45 to 30 ms, perhaps not long enough to reach steady state activation during a 50-ms test pulse. Since the activation of current can be fit with a single exponential, we extrapolated the fit to determine the conductance at steady state and recalculated the V1⁄2. We calculated that the apparent V1⁄2 in 1 $M calcium would be shifted no more than 9.3 mV positive by the slow activation time during the 50-ms test pulse, which was much less than the %29 mV G-V shift observed with H"4. " Subunits Alter Hslo1 Activation Kinetics—Fig. 6 shows the average activation time constants (%) versus voltage plots measured at 1, 10, and 50 $M calcium with representative traces measured in 1 $M calcium shown above each graph. At different calcium concentrations, Hslo1 channel activation rates (closed symbols) showed a similar voltage dependence, as indicated by similar slopes of the % activation/voltage curves. The effect of reducing calcium (different symbols) was to shift the % activation-voltage curve in a primarily parallel fashion to longer time constants. Our preliminary observation of Hslo1 currents with " subunits in 10 $M free calcium (Fig. 3) showed a significant slowing of activation kinetics by H"4, compared with the effects with other " subunits. As shown in Fig. 6D, the effect of H"4 in 10 $M free calcium was similar to reducing calcium concentration,

the % activation-voltage curve is shifted in a primarily parallel fashion to longer time constants without significant effects on the voltage dependence (slope). We looked in more detail over a range of calcium concentrations and found that at a low calcium concentration (1 $M free calcium), M"1 and H"2! also slowed activation rates and to similar rates as H"4. This is most obvious in the traces of representative currents (shown above %-voltage plots) evoked by a 200-mV step in 1 $M free calcium. Activation time courses could be fitted well with single exponential functions (fits are plotted overlapping current traces). M"1, H"2!, and H"4 slowed Hslo1 currents time constants from 2.7 & 0.30 to 10.27 & 1.9, 15.7 & 2.8, and 13.5 & 2.2 ms, respectively. At the intermediate calcium concentration of 10 $M, M"1 and H"2! subunits have no significant effect on Hslo1 activation kinetics. At high calcium (50 $M), M"1, H"2!, and H"4 have an apparent reduction in voltage dependence, seen as a shallower slope in the % versus voltage curve. In addition, M"1 slows activation. In contrast to other " subunits, H"3coexpressing channels show a small but consistent decrease in activation time constants at all calcium concentrations (Fig. 6C). " Subunit Effects on Hslo1 Deactivation Kinetics—We next measured the effects of the " subunits on deactivation kinetics of Hslo1 channels. Channels were maximally activated with a 160-mV depolarizing pulse and then stepped to different test


FIG. 6. Effect of ! subunits on Hslo1 activation kinetics. Plots of activation time constants as a function of voltage and calcium. Above each plot are shown representative current traces and single exponential fits (smooth trace) evoked by a 200-mV step in 1 $M free calcium. For comparison, the current traces were normalized to peak height. To measure activation rates, currents were elicited from $80 mV to more positive test potentials for 20 ms. Activation time courses were fit with a single exponential function % ' AeqFV/RT % b and visually inspected for a proper fit. Solid symbols show values for Hslo1 recordings alone and are duplicated in A–D for comparison to Hslo1 % " subunit recordings (open symbols) as indicated by the panel label. Recordings were made in internal solutions containing calcium concentrations of 1 $M, diamond symbols; 10 $M, square symbols; and 50 $M, circles. Error bars are S.E. of the means for n ' 6, 11, and 9 for Hslo1 alone; n ' 6, 6, and 5 for Hslo1 coexpressed with M"1; n ' 7, 8, and 3 for Hslo1 coexpressed with H"2!; n ' 9, 7, and 4 for Hslo1 coexpressed with H"3; n ' 11, 10, and 14 for Hslo1 coexpressed with H"4 (in 1, 10, and 50 $M calcium, respectively).

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voltages to measure deactivation time constants. Representative tail currents measured in 10 $M calcium are shown in Fig. 7A. Tail currents were well fit with single exponentials that are shown as dashed lines overlapping the traces. Time constants are plotted at different voltages in Fig. 7B or at $80 mV in different calcium concentrations in Fig. 7C. Hslo1 channels are voltage- and calcium-dependent in their relaxation of currents. Deactivation is slowed as the voltage is more positive (Fig. 7B, square symbols) until channels begin to activate at more positive tail potentials (not shown). The effect of increasing calcium is to slow deactivation and shift the %-voltage curves along the voltage axis without changing the slope (47). At 10 $M free calcium concentration M"1, H"2!, and H"4 had effects similar to increasing calcium. For example, the deactivation kinetics of Hslo1 were slowed, but the voltage dependence (slope) was not significantly affected (Fig. 7B). M"1 and H"2! had the most dramatic effect on Hslo1 deactivation kinetics (Fig. 7A), increasing the time constants from 0.42 & 0.02 to 7.2 & 0.49 and 11.2 & 1.8 ms, respectively (Fig. 7, B and C). Because calcium slows deactivation kinetics of Hslo1, it was surprising to find that H"4, which decreased the apparent calcium sensitivity of Hslo1 at low calcium concentrations, also slowed deactivation kinetics (% of 0.56 & 0.04 ms versus 0.2 & 0.01 ms in 1 $M

calcium concentration for H"4 and Hslo alone respectively, see Fig. 7, A and C). H"3 had no effect on deactivation at this or other calcium concentrations tested. DISCUSSION

The BK "1 subunit was first identified by copurification with slo ! subunits in tracheal smooth muscle (3). Subsequent characterization of "1/Hslo1 interaction showed that the "1 subunit affects Hslo1 channel pharmacology, gating kinetics, and calcium sensitivity (27). However, the restricted distribution of "1 subunits to smooth muscle posed the question of whether other " subunits are involved in modulating slo function in other tissues. The recent cloning of "2 (33, 34) and our results extend the BK " gene family to a minimum of four genes. The tissue distribution of the different " family members suggests that " subunits have a broad role in modulating slo channel. In addition, the overlapping tissue distribution creates the possibility that different " subunits coassemble with the same slo channel, further increasing diversity of BK channels. "1 and H"2 subunits share the most sequence similarity among the " subunits. With the exception of inactivation, we found these subunits appear to function similarly. Both subunits increase the apparent calcium sensitivity and slow chan-


FIG. 7. Effect of ! subunits on Hslo1 deactivation. Tail currents were evoked by a 10-ms step to 160 mV (for 10 $M or greater free calcium) or 200 mV (for 1 $M free calcium) and then measured by stepping down to various voltages for durations at least greater than four times the deactivation time constants. A, representative tail currents measured at $80, $60, $40, $20, 0, and %10 mV. Currents were fit well by single exponential functions (% ' AeqFV/RT % b) and are shown by the dashed traces. B, plots of deactivation time constants versus voltage measured in 10 $M free calcium. Note: Hslo1 (closed square) and H"3 (open square) data points are overlapping and difficult to distinguish. Data points reflect means deactivation time constants & S.E. for n ' 10 for Hslo1 alone; n ' 10 for Hslo1 with M"1; n ' 8 for Hslo1 with H"2!; n ' 8 for Hslo1 with H"3; n ' 10 for Hslo1 with H"4. C, plots of deactivation time constants measured at $80 mV and in various internal calcium concentrations. Error bars are S.E. for n ' 7, 5, 4, and 6 for Hslo1 alone; n ' 5 and 10 for Hslo1 with M"1; n ' 8 and 8 for Hslo1 with H"2!; n ' 8, 4, and 5 for Hslo1 with H"3; n ' 9, 10, 9, and 7 for Hslo1 with H"4 (in 1 and 10 $M calcium, respectively, for M"1 and H"2! or 1, 10, 50, and 200 $M calcium, respectively, for Hslo1 alone, Hslo1 with H"3, and Hslo1 with H"4).

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Acknowledgments—We gratefully acknowledge L. Toro for providing the Hslo1 cDNA expression construct. We also thank C. L. Hogan and F. T. Horrigan for comments on this manuscript. REFERENCES 1. Knaus, H. G., Garcia-Calvo, M., Kaczorowski, G. J., and Garcia, M. L. (1994) J. Biol. Chem. 269, 3921–3924 2. Garcia-Calvo, M., Knaus, H. G., McManus, O. B., Giangiacomo, K. M., Kaczorowski, G. J., and Garcia, M. L. (1994) J. Biol. Chem. 269, 676 – 682 3. Knaus, H. G., Folander, K., Garcia-Calvo, M., Garcia, M. L., Kaczorowski, G. J., Smith, M., and Swanson, R. (1994) J. Biol. Chem. 269, 17274 –17278 4. Tanaka, Y., Meera, P., Song, M., Knaus, H. G., and Toro, L. (1997) J. Physiol. (Lond.) 502, 545–557 5. Marrion, N. V., and Tavalin, S. J. (1998) Nature 395, 900 –905 6. Prakriya, M., and Lingle, C. J. (1999) J. Neurophysiol. 81, 2267–2278 7. Sah, P. (1996) Trends Neurosci. 19, 150 –154 8. Poolos, N. P., and Johnston, D. (1999) J. Neurosci. 19, 5205–5212 9. 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(1998) Proc. R. Soc. Lond. Ser. B. Biol. Sci. 265, 685– 692 19. Joiner, W. J., Tang, M. D., Wang, L. Y., Dworetzky, S. I., Boissard, C. G., Gan, L., Gribkoff, V. K., and Kaczmarek, L. K. (1998) Nat. Neurosci. 1, 462– 469 20. Toro, L., and Stefani, E. (1991) J. Bioenerg. Biomembr. 23, 561–576 21. Reinhart, P. H., and Levitan, I. B. (1995) J. Neurosci. 15, 4572– 4579 22. Sansom, S. C., Stockand, J. D., Hall, D., and Williams, B. (1997) J. Biol. Chem. 272, 9902–9906 23. DiChiara, T. J., and Reinhart, P. H. (1997) J. Neurosci. 17, 4942– 4955 24. Dworetzky, S. I., Boissard, C. G., Lum-Ragan, J. T., McKay, M. C., PostMunson, D. J., Trojnacki, J. T., Chang, C. P., and Gribkoff, V. K. (1996) J. Neurosci. 16, 4543– 4550 25. Tseng-Crank, J., Godinot, N., Johansen, T. E., Ahring, P. K., Strobaek, D., Mertz, R., Foster, C. D., Olesen, S. P., and Reinhart, P. H. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9200 –9205 26. Meera, P., Wallner, M., Jiang, Z., and Toro, L. 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nel gating to similar extents. H"3 and H"4 are more distantly related on the sequence level and functionally dissimilar as well. H"3 speeds activation kinetics, whereas H"4 slows gating kinetics and has a complex effect on the apparent calcium sensitivity. The mechanism of " subunit modulation of slo channels has not been extensively studied. Single channel analysis of slo coexpressed with bovine "1 subunits shows that " subunits increase open probability by stabilizing the channel in a bursting state, perhaps by increasing an energetic barrier for leaving the open state (29). Two functional consequences are predicted from these results. The first is that slo/"1 channels have an increased open probability at equivalent voltages compared with slo channels alone. The increased open probability is seen as an apparent increased calcium sensitivity or perhaps more accurately described as a negative shift in the V1⁄2 (since it is not clear whether "1 subunits affect calcium affinity directly). The second is that channels should deactivate more slowly. For M"1 and H"2!, and H"4 at high calcium concentrations, this appears to be the case. However, for H"4 at low calcium concentrations, the negative shift in the V1⁄2 and slowed gating kinetics were not correlated, that is at low calcium H"4 slowed deactivation kinetics while shifting the V1⁄2 to more positive potentials. Assuming that the mechanism by which the different " subunits affect Hslo1 activation, deactivation, or conductance-voltage relationship are conserved, then one explanation would be that " subunits have some effects on Hslo1 channel gating kinetics that are not coupled to the effects observed for steady state conductance-voltage relationships. The difference in the properties of the different " subunits should provide useful tools in molecularly characterizing the structures of the " subunits that confer modulation of slo channels. An interesting observation was that " subunit modulation of Hslo1 channels was, in many aspects, not a uniform shifting of gating time or half-activation voltage but was tuned to calcium concentrations. For example, the effect of H"4 on the V1⁄2 was a shift either to positive or negative membrane potentials, depending on the calcium concentrations (Fig. 5E). In addition, M"1, H"2, and H"4 slow activation kinetics at low but not at high calcium concentrations (Fig. 6, A and B). The tuning of ! subunit function could be important in different cell types or subcellular locales where the resident " subunit could appropriately modulate BK channel properties to the cellular environment. In nerve terminals, where BK channels are associated with calcium channels (10, 48) the calcium microenvironment can vary from 10 to 100 $M during calcium influx through calcium channels (49). H"4 would be expected to steepen the response of BK channels to calcium changes, suppressing BK channels at low calcium, but enhancing BK channel openings as calcium levels reach threshold concentrations. BK channels have been characterized in tissues by differences observed in unitary conductance, gating kinetics, apparent calcium sensitivity, and pharmacology (50). Much of the differences in electrophysiological properties can be accounted for by the extensive alternative splicing that the slowpoke genes undergo (14 –18) and the effects caused by phosphorylation of the channel (20 –22). We have demonstrated that a great deal of BK channel diversity is also produced by association with a large family of " subunits. Recent reports indicate that the extent of " subunit modulation of slo channels is dependent on the alternatively spliced slo variants interacting with the " subunit (31), further increasing the diversity possible. Future work should elucidate the effects of the " subunits on the physiology and pharmacology of the slo channels and allow us to ascribe function of the cloned " subunits to the diverse BK channels observed in situ.