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J. Biol. Chem., Vol. 277, Issue 25, 22119-22122, June 21, 2002

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ACCELERATED PUBLICATION
The Large Conductance Potassium Channel -Subunit Can Interact with and Modulate the Functional Properties of a Calcium-activated Chloride Channel, CLCA1^*

Iain A. Greenwood, Lisa J. Miller, Susumu Ohya, and Burton Horowitz^§

From the Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557-0046

Received for publication, April 5, 2002, and in revised form, April 19, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have recently compared the biophysical and pharmacological properties of native Ca²⁺-activated Cl currents in murine portal vein with mCLCA1 channels cloned from murine portal vein myocytes (Britton, F. C., Ohya, S., Horowitz, B., and Greenwood, I. A. (2002) J. Physiol. (Lond.) 539, 107-117). These channels shared a similar relative permeability to various anions, but the expressed channel current lacked the marked time dependence of the native current. In addition, the expressed channel showed a lower Ca²⁺ sensitivity than the native channel. As non-pore-forming regulatory -subunits alter the kinetics and increase the Ca²⁺ sensitivity of Ca²⁺-dependent K⁺ channels (BK channels) we investigated whether co-expression of -subunits with CLCA1 would alter the kinetics/Ca²⁺ sensitivity of mCLCA1. Internal dialysis of human embryonic kidney cells stably expressing CLCA1 with 500 nM Ca²⁺ evoked a significantly larger current when the -subunit KCNMB1 was co-expressed. In a small number of co-transfected cells marked time dependence to the activation kinetics was observed. Interaction studies using the mammalian two-hybrid technique demonstrated a physical association between CLCA1 and KCNMB1 when co-expressed in human embryonic kidney cells. These data suggest that activation of CLCA1 can be modified by accessory subunits.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In various cell types including smooth muscle, secretory, and endothelial cells chloride currents activated by an increase in intracellular calcium over 150 nM (termed I_Cl.Ca) exist that exhibit distinctive voltage- and time-dependent characteristics (1, 2). However, little is known about the molecular identity of the channels underlying I_Cl.Ca, although the CLCA gene family has been proposed to encode for calcium-sensitive chloride channels (3-6). We showed recently that mouse portal vein smooth muscle cells, which exhibit a robust Ca²⁺-activated Cl current, expressed mCLCA1 but not mCLCA3 (7). Expression of this gene in human embryonic kidney (HEK)¹ cells generated a chloride current that required considerably higher concentrations of Ca²⁺ for activation (2 mM) than native I_Cl.Ca. Moreover the current generated did not exhibit the time- and voltage-dependent kinetics of the native channels in this cell type (7). Non-pore-forming auxiliary or -subunits alter K⁺ channel kinetics and increase the Ca²⁺ sensitivity of large conductance K⁺ channels (BK channels) (8). Consequently the aim of the present study was to determine whether co-expression of an auxiliary -subunit encoded by the KCNMB1 gene with mCLCA1 could result in Cl currents with time-dependent characteristics and Ca²⁺ sensitivity similar to the native I_Cl.Ca. While marked differences between the native current and the current produced by co-expression of mCLCA1 and KCNMB1 still existed the results of the present study show that co-expression of a -subunit shown to modulate K⁺ channel activity augmented the amplitude of Cl currents generated by the expression of mCLCA1. In addition, a direct interaction of the proteins was determined using a mammalian two-hybrid assay.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Molecular Biological Methods-- Membrane currents were recorded from HEK cells that stably expressed mCLCA1. The full lengths of murine CLCA1 (mCLCA1) and KCNMB1 (mKCNMB1) were ligated into mammalian expression vectors, pcDNA3.1 and pZeoSV (Invitrogen), respectively. After cloning, HEK293 cells were transiently transfected with mCLCA1 by calcium phosphate precipitation. Cells were then incubated in medium containing 1% geneticin (Invitrogen) to select for transfected cells (7). Recordings were made from HEK cells that expressed mCLCA1 only (termed control) or co-expressed with KCNMB1, which generates a non-pore-forming subunit that increases K⁺ channel Ca²⁺ sensitivity. Expression of KCNMB1 and mCLCA1 was checked by RT-PCR. Total RNA was isolated from individual HEK cells using the SNAP Total RNA isolation kit (Invitrogen) following the manufacturer's instructions, including the use of polyinosinic acid (20 µg) as an RNA carrier. First-strand cDNA was prepared from the RNA using the Superscript II^TM Reverse Transcriptase kit (Invitrogen). 1 µg of total RNA was reverse-transcribed with 200 units of reverse transcriptase in a 20-µl reaction containing 25 ng of oligo(dT)_12-18 primer, 500 µM each dNTP, 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl₂, and 10 mM dithiothreitol. PCR was performed with gene-specific primers for mCLCA1, mKCNMB1, and -actin using AmpliTaq Gold reagents (Applied Biosystems, Foster City, CA). The following PCR primers (GenBank^TM accession numbers in parentheses) were used: mCLCA1 (AF047838), sense nt 1781-1800 and antisense nt 1868-1886, amplicon = 105 bp; mKCNMB1 (AF020711), sense nt 443-466 and antisense nt 571-593, amplicon = 151 bp. The amplification profile for these primer pairs were as follows: a 10-s denaturation step at 95 °C, a 10-s annealing step at 60 °C, and a 30-s primer extension step at 72 °C for 25 and 35 cycles for mCLCA1 and mKCNMB1, respectively, performed in a GeneAmp 2400 thermal cycler (Applied Biosystems). RT-PCR with -actin primers controlled for genomic DNA contamination in the source RNA since these primers were designed to span two exons and an intron. The amplified products were separated by electrophoresis on a 2% agarose/1× TAE (Tris, acetic acid, EDTA) gel, and the DNA bands were visualized by ethidium bromide staining. The no-template control was a PCR amplification for which the template was not added, controlling for nonspecific amplification and spurious primer-dimer fragments. Each amplified product was sequenced by the chain termination method with an ABI PRIZM DNA sequencer model 310 (Applied Biosystems).

Solutions-- The external solution used to bathe the cells had the following composition: 126 mM NaCl, 10 mM HEPES, 20 mM glucose, 1.8 mM CaCl₂, 1.2 mM MgCl₂, 10 mM triethanolamine hydrochloride, and the pH was set to 7.2 with 10 M NaOH. The pipette solution used to activate Cl currents was the same as that used to study native I_Cl.Ca in murine myocytes (7) and contained 20 mM triethanolamine hydrochloride, 106 mM CsCl, 5 mM HEPES, 10 mM BAPTA, 3 mM MgATP, 0.2 mM GTP disodium, and 0.42 mM MgCl₂, and the pH was set to 7.2 with CsOH. [Ca²⁺] was buffered by adding the appropriate amount of CaCl₂ determined by the EQCAL buffer program (Biosoft, Ferguson, MO). Currents elicited by 2 mM Ca²⁺ were recorded with a pipette solution described by Gandhi et al. (5) and Gruber et al. (6) that contained mM 126 N-methyl-D-glucamine chloride, 30 mM sucrose, 5 mM HEPES, 2 mM MgCl₂, and 2 mM CaCl₂. In all experiments control cells were alternated with cells transiently transfected with KCNMB1 to ensure that the data were obtained under the same experimental conditions. Cells were held at 60 mV, and current-voltage relationships were constructed by stepping to test potentials between 100 and +120 mV for 1.5 s. The relative permeability of the more permeable anion SCN and the less permeable anion isethionate was determined by applying the Goldman-Hodgkin-Katz equation to the shift in reversal potential produced by replacement of the normal external solution for one containing equimolar concentrations of either NaSCN or sodium isethionate instead of NaCl (see Ref. 9 for a better description). Changes in junction potential were minimized by the use of 300 mM KCl agar bridge. All data are reported as the mean of n cells ± S.E.

Mammalian Two-hybrid Assay-- HEK cells from a 75-cm² flask at 80% confluence (approximately 3 × 10⁶ cells) were electroporated at 300 V, 25 mA, 25 watts, 1000 µF with 4 µg of pG5CAT plasmid and 20 µg of each plasmid of interest. The transfected cells were plated out in a 25-cm² flask and grown for 48 h. At 48 h, the cultures were lysed with reagents supplied with the chloramphenicol acetyltransferase (CAT) enzyme-linked immunosorbent assay kit (Roche Molecular Biochemicals) according to the manufacturer's protocol, frozen in liquid nitrogen, and stored at 80 °C until sufficient samples had been accumulated to perform a full assay with positive and negative controls. The CAT enzyme-linked immunosorbent assay was performed according to the manufacturer's protocols with no alterations and read on a Molecular Devices (Sunnyvale, CA) plate reader to determine CAT concentration in each sample. A portion of each sample was assayed by spectrophotometry with the Bio-Rad DC Protein Assay according to the manufacturer's protocol to determine total protein concentration.

The A₄₀₅ from the CAT assay was divided by the A₇₅₀ from the Lowry total protein assay to determine a ratio of CAT to total protein. This ratio was then compared with the ratio for the negative controls to give a -fold increase of CAT expression. The negative control for this experiment was a transfection with -cslo in plasmid pM (20 µg) plus pG5CAT (4 µg). The positive control supplied by the manufacturer was pM3-VP16 (20 µg) plus pG5CAT (4 µg). However, an additional positive control was generated by cloning the -cslo gene into pVP16 and co-transfecting with -cslo in pM (20 µg). The specific interaction between mClCA1 and -cslo was determined from transfection with -cslo in pM (20 µg) and mClCA1 in pVP16 (20 µg) plus pG5CAT (4 µg). All assays were repeated three times to determine the reproducibility of the assay.

Reagents-- All enzymes and drugs were obtained from Sigma.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

RT-PCR using primers specific to the individual genes determined the expression of mCLCA1 and KCNMB1 in HEK 293 cells. Fig. 1Ai shows that in untransfected HEK cells there was no detectable expression of either mCLCA1 or KCNMB1. In comparison, mCLCA1 amplicons were detected in HEK cells stably expressing mCLCA1 alone and in the presence of KCNMB1, and notable expression was detected for KCNMB1 only in those HEK cells transiently transfected with KCNMB1. Actin primers were used to confirm that the products generated were representative of RNA (498 bp) and not contaminated with genomic DNA (intron containing 708-bp band) because these primers were designed to span an intron as well as two exons. This control serves the identical purpose as a cDNA reaction lacking reverse transcriptase; however, it can be performed on the same RNA preparation as the test reactions.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Ca2+-activated currents in HEK cells expressing mCLCA1. Ai, RT-PCR detection of mCLCA1 and mKCNMB1 in native, mCLCA1-expressed, and mCLCA1/mKCNMB1-co-expressed HEK293 cells: HEK-C, HEK-CLCA1, and HEK-CLCA1/KCNMB1, respectively. PCR products were generated through the use of gene-specific primers for murine CLCA1 and KCNMB1. Amplified products were separated on 2.0% agarose gels and were identified by ethidium bromide staining. A 100-bp molecular weight marker was used to estimate the size of the amplicon, and the migration is shown on the right. RT-PCR performed in the presence of -actin gene-specific primers demonstrates that the products are representative of RNA (498 bp, see "Experimental Procedures"). Aii and Aiii show an ensemble of currents evoked by 500 nM Ca2+ in HEK cells expressing mCLCA1 alone (Aii) or co-expressing KCNMB1 (Aiii). B shows the mean late current evoked by 500 nM Ca2+ in cells expressing mCLCA1 alone (solid symbols, n = 12) or mCLCA1 + KCNMB1 (open squares, n = 17). Currents elicited by 250 nM Ca2+ in HEK cells expressing both genes are shown by open triangles (n = 9). Late current was recorded at the end of the test step immediately prior to repolarization to the holding potential of 50 mV. Di and Dii show a comparison of the kinetics of ICl.Ca from one of five of the HEK cells expressing mCLCA1 and KCNMB1 that displayed time dependence to the activation and deactivation (Di) with native ICl.Ca recorded from murine portal vein myocytes (Dii). Currents were generated by pipette solutions containing 500 nM Ca2+, and currents were recorded at test potentials between 100 and +120 mV from a holding potential of 50 mV. Currents with outward relaxations at potentials positive of +60 mV were observed in 5 of 17 cells expressing mCLCA1 + KCNMB1.

Effect of KCNMB1 Expression on mCLCA1 Currents-- In our previous study we observed that mCLCA1 cloned from murine portal vein when expressed in HEK cells generated Cl currents that did not exhibit any time dependence at potentials between 100 and +100 mV and that required a high Ca²⁺ concentration (2 mM) for appreciable current to be generated (7). This was similar to previous expression studies on CLCA genes (5, 6). Identical relatively Ca²⁺-insensitive currents that lacked any time dependence were recorded from control cells (mCLCA1 only) in the present study (Fig. 1Aii). The lack of time-dependent kinetics upon stepping to positive test potentials was not due to a saturation of channel activation with the high Ca²⁺ as the small currents generated by 500 nM Ca²⁺ in the pipette also showed no time dependence (see Fig. 1Aii for example). When currents were recorded from cells expressing both mCLCA1 and KCNMB1 there was a significant increase in the amplitude of current recorded (Fig. 1, Aiii and B). Thus, the instantaneous current evoked by a 500 nM Ca²⁺ pipette solution at +80 mV in cells that co-expressed mCLCA1 and KCNMB1 was 20 ± 4 pA pF¹ (n = 16) compared with 3.7 ± 0.8 pA pF¹ in cells expressing mCLCA1 alone (n = 12, Fig. 1B). In addition, co-expression of KCNMB1 with mCLCA1 augmented markedly the inward current compared with control cells (mean amplitude at 80 mV was 1.9 ± 0.3 and 8.6 ± 2 pA pF¹ for control and test cells, respectively). Dialysis of HEK cells co-expressing mCLCA1 and KCNMB1 with a pipette solution containing 250 nM Ca²⁺ also produced currents significantly larger than those produced by 500 nM Ca²⁺ in cells expressing mCLCA1 alone (Fig. 1, B and C). Moreover the currents activated by 500 nM Ca²⁺ in cells expressing mCLCA1 and KCNMB1 were significantly larger than currents evoked by 2 mM in HEK cells expressing mCLCA1 alone (Fig. 1C).

In all (18/18) of the HEK cells expressing mCLCA1 alone the currents elicited by 500 nM Ca²⁺ showed no time dependence unlike native I_Cl.Ca. When HEK cells were transfected with mCLCA1 and KCNMB1, 5 of 17 cells exhibited some time dependence at potentials greater than +60 mV (see Fig. 1Di for example). However, it is worth noting that in these cells the kinetics of the inward relaxation was significantly faster than those of native I_Cl.Ca (see Ref. 7 and Fig. 1Dii for comparison). In those five HEK cells that exhibited an outward relaxation at +80 mV the mean time constant for the exponential fit to this relaxation was 142 ± 29 ms, and the mean time constant for the consequent inward current at 60 mV was 32 ± 6 ms. These values are considerably faster than those recorded for native I_Cl.Ca in murine portal vein cells where mean values at +80 and 60 mV are about 300 and 80 ms, respectively (7). Thus, co-expression of KCNMB1 with mCLCA1 enhances the amplitude of Ca²⁺-activated currents in HEK cells. However, the characteristic kinetics of the native current was not reproduced fully by the co-expression of the auxiliary subunit with mCLCA1.

Ionic Nature of Current Evoked in HEK Cells Co-expressing KCNMB1 and mCLCA1-- It is possible that the increased amplitude of the Ca²⁺-activated current produced by the co-expression of KCNMB1 and mCLCA1 was due to contamination from another ionic conductance. We therefore performed anion replacement studies to confirm that the augmented current in the doubly transfected HEK cells was a Cl current. We showed previously that replacement of external NaCl with an equimolar concentration of NaSCN produced an approximately 45-mV shift of the reversal potential (E_rev) of the current generated by mCLCA1 expression (7). This resulted in a permeability of SCN relative to Cl (P_SCN/P_Cl) of about 5.8 (7). Replacement of the external anion with the more permeant SCN produced a marked leftward shift in the current-voltage relationship of the current produced by 500 nM Ca²⁺ in HEK cells expressing both mCLCA1 and KCNMB1. Consequently the mean E_rev changed from 1.2 ± 2 mV in NaCl to 49.5 ± 5 mV in NaSCN (n = 5). This resulted in a mean P_SCN/P_Cl of 7.48 ± 1.3 (n = 5) that was not significantly different from the P_SCN/P_Cl calculated for currents produced by mCLCA1 expression alone (5.88 ± 0.95, n = 5). The current generated by the co-expression of mCLCA1 and KCNMB1 was rapidly and markedly inhibited by the Cl channel blocker niflumic acid, similar to previous reports on the effects of this agent on mCLCA1 (6). Application of 100 µM niflumic acid for 3 min reduced the recorded current at +80 mV from 152 ± 25 to 37 ± 12 pA (n = 3). These data show that the current generated by co-expression of mCLCA1 and KCNMB1 shares identical pharmacological and ionic selectivity properties as the current generated by mCLCA1 expression alone.

Mammalian Two-hybrid Experiments-- We have shown that KCNMB1 co-expression with mCLCA1 can modulate the functional properties of the channel. We were interested in whether these effects were due to a direct interaction between the proteins or through another intermediary protein. We used a mammalian two-hybrid system to test full-length proteins in the same mammalian expression system that we used for functional expression (10-12). KCNMB1 was expressed as a fusion protein to the GAL4 DNA-binding domain in plasmid pM. CLCA1 was expressed as a fusion protein to the GAL4 activation domain in plasmid pVP16. pG5CAT was used as the reporter vector, which contains the CAT gene downstream of five consensus GAL4 binding sites and the minimal promoter of the adenovirus E1b gene (Clonetech Laboratories, Palo Alto, CA). The negative control used to normalize the experiments was a co-transfection of pM--cslo and pG5CAT. CAT activity for these cells was set at 1, and levels for the remaining transfections were relative to the negative control. The positive control provided by Clonetech (pM3-VP16) expresses a fusion of the GAL4 DNA-binding domain to the VP16 activation domain. When co-expressed with pG5CAT, this construct yielded CAT activity 2.63 ± 0.09-fold (n = 4, Fig. 2) above the negative control. This value is similar to that reported by other investigators (11). When we co-transfected pVP16-mCLCA1 and pM--cslo and the reporter pG5CAT we observed a dramatic increase in CAT activity of 33.7 ± 3.3-fold (n = 4, Fig. 2) above the negative control. As a natural positive control we used pVP16--cslo and pM--cslo and the reporter pG5CAT. This combination yielded a CAT activity of 7.9 ± 0.6 (n = 3, Fig. 2).

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Mammalian two-hybrid data. Transient transfection was conducted in HEK293 cells using pG5CAT as reporter. -Fold activity is compared with base-line activation (pG5CAT + -cslo in pM) as determined by the amount of CAT expressed in transfections with -cslo in pM alone. The x axis indicates the plasmids transfected in each experiment. The y axis measures -fold activity.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study we have tested the hypothesis that mCLCA1 could combine with auxiliary subunits normally associated with BK channels to modulate its functional properties. The CLCA family of chloride channels shares a number of similarities with I_Cl.Ca recorded from epithelial cells (14) but is less similar to I_Cl.Ca recorded from smooth muscle cells (1, 15), cardiac myocytes (16), or endothelial cells (17). Therefore, either mCLCA1 does not encode I_Cl.Ca in these cell types, or auxiliary subunits are required to modulate the activity of mCLCA1. In the present study we show that mCLCA1 can associate with the BK -subunit to increase its sensitivity to [Ca²⁺]_i and in a small number of cells appeared to alter the kinetics of activation and deactivation. However, we also show that I_Cl.Ca recorded in native portal vein from mice displays some different kinetics than that of mCLCA1 with BK -subunit. Therefore, while the BK -subunit can associate with mCLCA1, it may not be used in reconstituting I_Cl.Ca in portal vein myocytes.

This is the first report of an auxiliary subunit interacting with a CLCA channel, and from our studies it is not understood where on the channel this interaction takes place. In vitro translation of CLCA homologs results in the synthesis of two proteins that are cleavage products of a single translated protein (14). For mCLCA1, the products are 90- and 32-38-kDa peptides with the smaller peptide derived from the carboxyl terminus (5). The functional relationship between the two peptides is not clear; however, the smaller peptide is not required for CLCA channel function (19). The predicted topological structure for the CLCA channels depicts five transmembrane-spanning domains containing a large extracellular amino terminus with several potential glycosylation sites (18). The cleavage site releases the carboxyl terminus along with two of the transmembrane domains. No data are available that would suggest a region of the protein involved in anion permeation, and it is not known whether the cleaved carboxyl terminus combines with the remainder of the protein to form a channel with five transmembrane segments. Future experiments using the two-hybrid system will aim to identify specific domains on mCLCA1 and KCNMB1 that are involved in the interaction.

A recent study has identified a new member of the CLCA gene family (mCLCA4) that is predominantly expressed in smooth muscles (13). The amino acid sequence for mCLCA4 is similar to mCLCA1 (79% identity), and few electrophysiological properties were reported by Elble et al. (13). The current was activated by ionomycin with 1.8 mM Ca²⁺ present in the bath solution and by the application of methacholine to cells expressing mCLCA4. It is not clear from Elble et al. (13) of the [Ca²⁺]_i required to elicit currents, and no pharmacology was performed on the expressed currents. Moreover the evoked currents generated by the expression of mCLCA4 exhibited no time-dependent activation at positive potentials, similar to currents produced by mCLCA1 expression (7). Therefore, mCLCA4 currents may be quite similar to mCLCA1 currents when expressed alone and dissimilar to native I_Cl.Ca. We have performed RT-PCR with primers specific for mCLCA4 (similar to those used by Elble et al. (13)) and also demonstrated robust expression in gastrointestinal smooth muscles; however, we could not detect mCLCA4 expression in murine portal vein.² Therefore, mCLCA4 may underlie I_Cl.Ca in some smooth muscles but not in this preparation, and it remains to be seen whether the properties of mCLCA4 mimic the smooth muscle current.

In summary, we have shown that auxiliary subunits such as the BK -subunit can associate and modulate mCLCA1 functional properties. The currents generated by the co-expression of the BK -subunit with mCLCA1 have Ca²⁺ sensitivity more typical of native I_Cl.Ca in murine portal vein myocytes. However, the majority of currents generated by the co-expression of mCLCA1 and mKCNMB1 did not exhibit the distinctive voltage-dependent kinetics that are a characteristic of the native I_Cl.Ca in smooth muscle cells. Since co-expressing mCLCA1 and mKCNMB1 failed to fully reconstitute I_Cl.Ca currents, another molecular candidate/auxiliary subunit may also associate with CLCA products in native preparations. Alternatively CLCA genes may not encode for the native chloride channel protein, and the true chloride channel gene underlying I_Cl.Ca in smooth muscle remains to be identified.

	ACKNOWLEDGEMENTS

We thank Martha Baring and Heather Beck for excellent technical assistance.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants DK 41315, HL 49254, and P20 RR15581.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

A Wellcome Trust research fellow.

^§ To whom reprint requests should be addressed: Dept. of Physiology and Cell Biology, University of Nevada School of Medicine, MS352/Anderson Medical Bldg., Reno, NV 89557-0046. Tel.: 775-784-1462; Fax: 775-784-6903; E-mail: burt@physio.unr.edu.

Published, JBC Papers in Press, May 6, 2002, DOI 10.1074/jbc.C200215200

² I. A. Greenwood, L. J. Miller, S. Ohya, and B. Horowitz, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: HEK, human embryonic kidney; BK channel, Ca²⁺-dependent K⁺ channel; CLCA, Ca²⁺-activated chloride channel; m, murine; RT, reverse transcription; nt, nucleotides; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; F, farads; CAT, chloramphenicol acetyltransferase.

	REFERENCES

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