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iridectomy scissors. The tectorial membrane was removed with a fine needle. The basilar papilla was collected with a fine bore suction pipette. Each tissue type ...
cSlo encodes calcium-activated potassium channels in the chick’s cochlea G.-J. J I A N G", M. Z I D A N I C", R. L. M I C H A E L S#, T. H. M I C H A E L", C. G R I G U E R#†    P. A. F U C H S"* " The Center for Hearing Sciences, Department of Otolar’ngolog’, Head and Neck Surger’, Johns Hopkins UniŠersit’ School of Medicine, Baltimore, MD, USA # Department of Ph’siolog’, UniŠersit’ of Colorado School of Medicine, DenŠer, CO, USA

SUMMARY

Large conductance, calcium-activated (BK) potassium channels play a central role in the excitability of cochlear hair cells. In mammalian brains, one class of these channels, termed Slo, is encoded by homologues of the Drosophila ‘ sloWpoke ’ gene. By homology screening with mouse Slo cDNA, we have isolated a fulllength clone (cSlo1) from a chick’s cochlear cDNA library. cSlo1 had greater than 90 % identity with mouse Slo at the amino acid level, and was even better matched to a human brain Slo at the amino and carboxy termini. cSlo1 had none of the additional exons found in splice variants from mammalian brain. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to show expression of cSlo1 in the microdissected hair cell epithelium (basilar papilla). Transient transfection of HEK 293 cells demonstrated that cSlo1 encoded a potassium channel whose conductance averaged 224 pS at ­60 mV in symmetrical 140 mM K+. Macroscopic currents through cSlo1 channels were blocked by scorpion toxin or tetraethyl ammonium, and were voltage and calcium dependent. cSlo1 is likely to encode BK-type calcium-activated potassium channels in cochlear hair cells.

et al. 1992). Homologous genes have been identified in mammals (Butler et al. 1993 ; Tseng-Crank et al. 1994). The Slo gene encodes a channel containing six transmembrane domains and a pore region that are homologous to the equivalent portions of voltage-gated potassium channels. In addition, Slo encodes an extended carboxy domain that may contain additional transmembrane segments, and is thought to encompass the calcium activation function of the channel (Wei et al. 1994). Slo transcripts in flies and mammals are subject to alternative splicing, resulting in pronounced variability of the extended carboxy segment of the channels. Some splice variants have altered gating kinetics (Lagrutta et al. 1994), suggesting a possible mechanism for generating the functional heterogeneity seen in hair cell BK currents. A second (beta) subunit can combine with the channel (Knaus et al. 1994) and could also alter function (McManus et al. 1995 ; Dworetzky et al. 1996). Already, the chick’s basilar papilla has proven valuable for the study of transcriptional regulation of voltage-gated ion channels in hair cells (Navaratnam et al. 1995). The expression of BK channels in chick cochlear hair cells varies both topologically and developmentally (Fuchs et al. 1988 ; Fuchs & Sokolowski 1990), prompting this search for BK channel genes whose regulation underlies these patterns of expression. We have used a partial clone of mouse Slo to perform homology screening of a chick’s

1. I N T R O D U C T I O N

The response dynamics of mechanosensory hair cells depend on the number and kinds of potassium channels found in each cell. For example, large conductance, calcium-activated (BK) potassium channels support electrical tuning in hair cells of frogs (Lewis & Hudspeth 1983 ; Hudspeth & Lewis 1988), turtles (Art & Fettiplace 1987), alligators (Fuchs & Evans 1988) and chickens (Fuchs et al. 1988). The electrical resonant frequency depends on the number and kinetics of BK channels in each electrically tuned hair cell (Art & Fettiplace 1987 ; Art et al. 1995). Further, the appearance of BK channels is coincident with a rapid stage of functional maturation during embryonic development of the chick’s cochlea (Fuchs & Sokolowski 1990). What structural differences amongst BK channels determine electrical tuning ? How are specific channel kinetics and numbers determined during development and maintained throughout the lifetime of the hair cell ? To begin to address these questions we sought to identify genes that may encode hair cell BK channels. A likely candidate is the Slo gene which encodes BK channels in Drosophila (Atkinson et al. 1991 ; Adelman * Author for correspondence (pfuchs!bme.jhu.edu) † Present address : VA Medical Center, Building 1, Rm 511, 1660 S. Columbian Way, Seattle, WA 98108-1532, USA. Proc. R. Soc. Lond. B (1997) 264, 731–737 Printed in Great Britain

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cochlear cDNA library. Here we report the identification and functional expression of a Slo homologue from the chick’s cochlea, cSlo1. Identification of a cDNA encoding calcium-activated potassium channels in hair cells provides new opportunities for the study of structure and function, as well as for regulation of this important class of membrane proteins. 2. M E T H O D S (a) Homology cloning from chick’s cochlear cDNA library Hatchling chicks (ranging from two days to two weeks in age) were decapitated and their external ear canals were opened tangentially. The cochlear duct was extracted through the widened oval window and frozen in liquid nitrogen within 1–2 min of decapitation. This method of dissection tears off the cochlear nerve and the great majority of neuronal somata. The remaining tissue includes the tegmentum vasculosum (analogous to the stria vascularis in mammalian cochlea), the lagena (a small otolithic organ in the cochlear apex), homogene cells, clear cells, hyaline cells and other surrounding cells, in addition to the hair cells and supporting cells of the basilar papilla (Oesterle et al. 1992). Five hundred frozen cochlear ducts (approximately 1 g wet weight) were used for construction of a cDNA library at Stratagene, Inc. (La Jolla, CA). cDNA was oligo-dT- and random-primed, size-selected (greater than 400 bp) and inserted into a ‘ Uni-Zap ’(Uni-ZapTM XR) vector system (Stratagene, Inc). The chick’s cochlear cDNA library contained 8.8 million plaque forming units (pfu) before amplification. The initial probe used for library screening was a 2.2 kb fragment of the mouse Slo (mSlo) cDNA that encoded a region of the channel including putative transmembrane regions S7 to S10 (provided by Dr L. Salkoff at Washington University). The probe was randomly labelled with $#PdCTP. Hybridization was carried out using recommended procedures (Stratagene, Inc.). After prehybridization at 42 °C for 4 h, hybridization proceeded overnight at 42 °C in 2¬Pipes (0.8 M NaCl, 0.02 M piperazine-N,N«-bis [2ethane-sulphonic acid] pH 6.5), 50 % de-ionized formamide, 0.5 % SDS and 100 µg}ml denatured, sonicated salmon sperm DNA. Filters were washed at room temperature in high salt before a final series of washes in 0.1¬‘ SSC ’ (15 mM NaCl, 1.5 mM Na-citrate, pH 7.0), 0.1 % SDS (sodium dodecyl sulphate) at 40–42 °C. Hybridizing clones were plaque-purified, amplified, and their inserts were analysed by Southern analysis with the same probe used for library screening. Positive inserts were subjected to restriction digest, subcloned, and sequenced (Sequenase 2.0TM, United States Biochemical, Cleveland, OH) in both directions until 100–200 bp of overlap were achieved. An ambiguous sequence was confirmed by repeated sequencing of different restriction products. (b) Reverse transcriptase polymerase chain reaction (RT-PCR) For dissection of specific tissue types, the cochlear duct was removed from hatchling chicks and treated with 0.1 mg}ml protease type XXIV (Sigma) for 10 min at room temperature. The cochlea was rinsed, and then immersed in DEPC-treated PBS (150 mM NaCl, 150 mM NaH PO , # % pH 7.4). The tegmentum vasculosum was cut free with iridectomy scissors. The tectorial membrane was removed with a fine needle. The basilar papilla was collected with a fine bore suction pipette. Each tissue type from one cochlear Proc. R. Soc. Lond. B (1997)

duct was collected in approximately 15 µl of PBS, added to 100 µl of Trizol lysis buffer (GibcoBRL) and triturated. Total RNA was extracted by treatment with chloroform and precipitation with isopropanol. Reverse transcription to cDNA was accomplished using a 20 µl mixture of buffer (50 mM KCl, 10 mM Tris-HCL, pH 8.3), 2.5 µM random hexamer primers, 0.25 mM each dNTP, 1.25 mM MgCl , 5 # U RNAse inhibitor and 25 U reverse transcriptase (Moloney murine leukaemia virus, GeneAmp kit, Perkin Elmer, New Jersey). The mixture was incubated at room temperature for 10 min, then at 42 °C for 45 min. The reaction was terminated by heating for 5 min at 96 °C, then placed on ice for 5 min. The 20 µl RT mixture was used as a template for PCR and added to 1.25 U Taq DNA polymerase and 10 pmoles of each primer in a total volume of 50 µl (same buffer as above). The forward and reverse primers for cSlo1 were : 5«TGTTTGCGAAACTAAAGC-3« (nucleotides 1829–1846), and 5«-GTGAGGTACAGTTCTGTA-3« (nucleotides 2876–2859). In parallel with PCR for cSlo1, another reaction was carried out with primers for chick β-actin as a control. The forward and reverse primers for chick β-actin were : 5«TGGATGATGATATTGCTGCG-3« (nucleotides 2–18), and 5«-CTCCATATCATCCCAGTTGG-3« (nucleotides 248–229). As a negative control, RNA samples that were not reverse transcribed were run in parallel. Thirty-five cycles of the PCR were performed with a Perkin Elmer 2400 Thermocycler : 94 °C for 20 s, 55 °C for 45 s, 72 °C for 90 s, followed by an additional 10 min at 72 °C for elongation. PCR products were analysed by electrophoresis in a 1 % agarose gel containing 0.5 µg}ml ethidium bromide. Detectable bands were obtained from as little as one-half of a basilar papilla. Amplification conditions for the primers were established initially with cSlo1 cDNA as a template, and then by amplifying cSlo1 from the cDNA library. Finally, RT-PCR reactions were run on chick brain. PCR products of the predicted size (1 kb) obtained from brain and whole cochlea were proven to be cSlo1 by sequencing. The RT-PCR products from brain or cochlear tissue were unlikely to arise from amplification of genomic DNA since that should have produced intronic sequences at the presumptive splice sites flanked by these primers. This conclusion was supported by the observation that treatment of the RNA extract with DNAase did not affect the RT-PCR products reported here. (c) Functional expression cSlo1 was subcloned into a eukaryotic expression vector (‘ pcDNA3 ’, Invitrogen, San Diego, CA) for expression in HEK 293 cells (human embryonic kidney cell line obtained from Dr D. Yue, Johns Hopkins University, Baltimore, MD). T-antigen DNA (pRSV T-antigen clone obtained from Dr R. Reed, Johns Hopkins University, Baltimore, MD) was included to enhance expression levels by activating the SV40 origin of replication in pcDNA3. A DNA}CaCl2 solution (final concentrations : 10 µg}ml cSlo1}pcDNA3, 20 µg}ml pBluescript as carrier, 2 µg}ml T-antigen clone and 62 mM CaCl ) was added by drops into an equal volume of 2X # HEPES- buffered saline (final concentrations : 140 mM NaCl, 25 mM N-[2-hydroxyethyl]piperazine-N«-[2ethane-sulphonic acid], 1.4 mM Na HPO , pH 7.1). HEK # % 293 cells grown on coverslips for 24 h were exposed to the precipitated DNA solution for 24 h, rinsed and returned to growth medium (DMEM 320–1965, 10 % fetal bovine serum, 1 % l-glutamine, 1 % penicillin} streptomycin and 0.1 % gentamicin, GIBCO). Coverslips were removed for recordings 24–72 h after transfection. ‘ Mock ’ (control) transfections were made by substituting cSlo1}pcDNA3 with an equivalent amount of carrier DNA (pBluescript), or by using a ‘ reverse

Cochlear Slo potassium channels G.-J. Jiang and others 733 insert ’ (3« to 5«) pcDNA3 clone of cSlo1. In later experiments, HEK 293 cells were co-transfected with green fluorescent protein (‘ pGreen Lantern-1 ’, Life Technologies, Gaithersburg, MD) as well as cSlo1. Transfected cells were identified by their bright green fluorescence when illuminated with 490 nm light, and in such cells the probability of recording cSlo1 channels approached unity. (i) Recording cSlo1 channel activity in HEK 293 cells was recorded in whole-cell, tight-seal, cell-attached and excised patch recordings with an Axopatch 1A (Axon Instruments, San Rafael, CA). Responses were filtered at 5 kHz and digitized, normally at 100 µs, for subsequent analysis. No leak subtraction was used. (ii) Experimental solutions For cell-attached, whole-cell and outside-out excised patch recording the bath solution consisted of 154 mM NaCl, 6 mM KCl, 5 mM CaCl , 2 mM MgCl , 5 mM HEPES, # # titrated to pH 7.4 with NaOH. The recording pipette was filled with an ‘ intracellular ’ solution containing 112 mM KCl, 30 mM KOH, 0.5 mM MgCl , 0.1 mM CaCl , 11 mM # # EGTA, 5 mM HEPES, titrated to pH 7.2. In some cases, intracellular calcium was buffered to 1–2 µM, as described below. For inside-out patches, the pipette contained 142 mM KCl, 0.5 mM MgCl , and 5 mM HEPES, pH 7.4. Following # formation of a cell-attached patch the bath solution was exchanged for one containing 142 mM KCl, 0.5 mM MgCl , # 5 mM HEPES, 1.08 mM CaCl , 2 mM EGTA, pH 7.2 (free # calcium being calculated to be 200 nM). Then a vesicle was excised and the outer membrane ruptured by cycling the patch through the air–water interface. To examine the effects of varying calcium concentration, 2 mM calcium buffer and varying amounts of calcium were added depending on the desired concentration of free calcium, as determined using MaxC software (Bers et al. 1994). In most cases, 2 mM Br BAPTA (Molecular Probes, Eugene, OR) and # 0.5–2.0 mM CaCl were combined to achieve free calcium # concentrations in the range of 0.5–50 µM. Lower levels of calcium were achieved using EGTA. The calcium activity of each solution was measured with a calcium electrode (MI600, Microelectrodes Inc., Bedford, NH.) which was calibrated using prepared standards (World Precision Instruments, Sarasota, FL). The scorpion venom peptides charybdotoxin (ChbTx– Peptides International, Louisville, KT) and iberiotoxin (IbTx) as well as tetraethyl ammonium (TEA) were dissolved in saline and added directly to the bath. Unless stated otherwise all chemicals were obtained from Sigma (St Louis, MO.). Recordings were made at room temperature (22–24 °C). Averaged results are given as mean³s.d. 3. R E S U L T S (a) The cSlo1 clone

A cDNA library derived from chick cochlear tissue was screened using a radio-labelled partial cDNA of mSlo. A number of hybridizing clones were plaquepurified by second and third screenings and the cDNA insert was characterized. A partial chick Slo clone was initially isolated (later named cSlo2, Genbank accession number U23823). It extends from amino acid 401 into the 3« untranslated region (UTR) and is homologous to mSlo1 (Butler et al. 1993 ; Genbank Proc. R. Soc. Lond. B (1997)

accession number L16912). To search for additional clones that might include more of the 5« coding region, the 5«-most 300 bp of the mSlo clone (not found in the chicken clone) was used as a probe for subsequent screening. This led to the isolation of a clone, subsequently named ‘ cSlo1 ’, containing a 3965 bp insert that covered the entire coding region. cSlo1 included an open reading frame of 3411 bp beginning 84 bp upstream of a putative start codon (based on homology to mammalian forms). The first stop codon was followed by 470 bp of 3« untranslated sequence. cSlo1 was described initially in abstract (Jiang et al. 1995) and has been assigned Genbank accession number U23821. The amino acid sequence predicted by the open reading frame is shown in figure 1. cSlo1 had greater than 90 % homology to the corresponding region of mouse brain Slo used in the initial screen (Butler et al. 1993). However, the entire coding region of cSlo1 was more similar to human brain Slo than to mouse forms. Further, cSlo1 had none of the additional exons that have been described at splice sites in mice and humans, making it equivalent in that respect to hbr1, the shortest human brain form (Tseng-Crank et al. 1994, Genbank accession number U11717). The overall identity of cSlo1 with hbr1 was 94 %. The variant amino acids in hbr1 are shown below their corresponding position in the cSlo1 sequence in figure 1. cSlo1 differed from hbr1 in having only four sequential serines upstream of the putative start codon, compared to 18 in the human sequence. Clusters of amino acid substitutions also were evident in the S1–S2 extracellular loop, and at the carboxy terminus.

(b) cSlo1 expression in basilar papilla

Reverse transcriptase polymerase chain reaction (RT-PCR) was used to demonstrate cSlo1 expression in basilar papilla. Total RNA isolated from microdissected basilar papilla was reverse transcribed to cDNA and then amplified with cSlo1 primers. Visible bands (stained with ethidium bromide) of the predicted size (1048 bp) could be obtained from a single basilar papilla (figure 2). Similar results were obtained in five additional experiments. RT-PCR from whole cochlear duct was run as a positive control. In addition to basilar papilla the microdissected tegmentum vasculosum was used for RT-PCR experiments. This secreting epithelium constitutes the bulk of the cellular mass of the cochlear duct (Cotanche & Sulik 1982 ; Oesterle et al. 1992). Here, too, a cSlo PCR product of 1 kb was found (figure 2). As a positive control, β-actin was also amplified from tegmentum vasculosum. RNA extracts that were not reacted with reverse transcriptase were used as negative controls and did not yield cSlo1 or β-actin PCR products. (c) Functional expression

As predicted by its homology to mouse and human Slo cDNAs, cSlo1 encoded large-conductance calciumactivated potassium channels. This was demonstrated

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Figure 1. Deduced amino acid sequence of cSlo1. Amino acids in hbr1 (human brain) that differ are shown below the cSlo1 sequence. Putative transmembrane domains labelled S1 through S10 and the pore region are demarcated by lines above the sequence. 1

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Figure 2. Reverse transcriptase polymerase chain reaction (RT-PCR) from cochlear tissue. Lane 1 : cSlo1 PCR product obtained from whole cochlear duct. Lane 2 : cSlo1 PCR product from microdissected basilar papilla. Lane 3 : cSlo1 PCR product from tegmentum vasculosum. Lane 4 : β-actin PCR product from tegmentum vasculosum. Approximate positions of the 1 kb and 0.3 kb size markers are indicated on the left. Non-RT controls were negative (not shown).

by transient transfection of HEK 293 cells. When cellattached patches in transfected cells were depolarized (voltage commands of ®100 to ®200 mV to the pipette) single channel currents of approximately 15 pA amplitude could be recorded (figure 3 a). The single channel conductance averaged 224³34 pS Proc. R. Soc. Lond. B (1997)

(chord conductance at ­50 to ­100 mV in 11 cellattached or inside-out patches with 140 mM extracellular potassium). Mock-transfected HEK 293 cells showed no evidence of large-conductance, calcium activated K+ channels. HEK 293 cells could express small conductance, voltage-gated (delayed rectifier) potassium channels, but total currents in excised patches from mocktransfected cells were negligible compared to those in cells expressing cSlo1. Also, the intrinsic delayed rectifier currents were insensitive to scorpion toxins, and activated at more negative membrane potentials than did cSlo1 currents when intracellular calcium was low. Transfected HEK 293 cells could express cSlo1 at very high levels. Indeed, single channel analyses were restricted by the rarity of patches with small numbers of channels. Thus cSlo1 channels have been characterized here largely by analysis of the ‘ macroscopic ’ currents across excised patches. Outward current across an outside-out patch depolarized to 0 mV is shown in figure 3 b (control). In this outside-out patch (2 µM free internal calcium), outward currents were activated positive to ®40 mV, rising to peak levels within several milliseconds. Tail currents decayed with a time constant of 2.5 ms after stepping back to ®40 mV. When 33 nM charybdotoxin (ChbTx) was added to the bath the outward currents were reduced by more than 90 %. Recovery from charybdotoxin block was variable but could be complete if the patch survived sufficient washing. In four experiments on outside-out patches, 33 nM ChbTx blocked 94 %³5.6 % of the

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current. In one whole-cell recording a ten-fold lower concentration of ChbTx (3.3 nM) blocked 32 % of the outward current. Iberiotoxin at 33 nM blocked 83 %³5.7 % of the current in five whole-cell recordings from transfected cells. Also, outward currents in transfected cells were sensitive to external tetraethyl ammonium (TEA). Approximately half (47 %³20 %, n ¯ 7) the outward current in whole-cell or outside-out patch recordings was blocked by 1 mM TEA. The calcium dependence of cSlo1 channels was determined using inside-out patches. Current amplitude at a given membrane potential increased with elevated cytoplasmic calcium (figure 4 a), demonstrating that the cSlo1 channels were calcium- as well as voltage-dependent. The rate of activation at ­100 mV increased while the deactivation rate of the tail currents at 0 mV was slower in higher calcium. The effect of calcium concentration on the steady-state voltagedependence of cSlo1 was determined by measurement of tail currents. These were normalized to the maximum (saturated) tail current, plotted as a function of the activation voltage and fitted with a Boltzmann function (figure 4 b). Steady-state activation was determined over a range of 200 mV at any one calcium concentration. The activation function of the channels Proc. R. Soc. Lond. B (1997)

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membrane potential (mV) Figure 4. (a) Current across inside-out patch during depolarization to ­100 mV. Tail currents at 0 mV. The holding potential was ®60 mV when calcium was 27 µM, ®50 mV for 1 µM, and ®20 mV for 0.1 µM calcium. Note that tail currents at 0 mV were larger and fell more slowly in elevated calcium. (b) Normalized tail current at 0 mV (ordinate) following voltage command plotted on abscissa. Voltage protocol as in panel (a). Smooth curves are Simplex fits of Boltzmann relation : I ¯ Imax}(1­exp[(V" ® # Vm)}K]). K ¯ RT}“F. V" was ®14 mV, 72 mV and 110 mV # for fits in 27, 1 and 0.1 µM calcium, respectively. K was 21 mV for all fits.

shifted to more negative voltages as calcium concentration rose. On average, V"? shifted 45 mV for a # ten-fold change in calcium concentration (from 26 Boltzmann fits on 16 excised patches over a range of calcium concentrations from 0.1 to 100 µM). 4. D I S C U S S I O N

cSlo1 from chick’s cochlea encoded a large conductance, calcium-activated potassium channel when expressed in HEK 293 cells. The single channel conductance of 224 pS was similar to that of channels encoded by human brain Slo, 200–250 pS (TsengCrank et al. 1994). Like other Slo homologues, cSlo1 channels were blocked by scorpion toxins (charybdo-

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toxin and iberiotoxin). The cSlo1 channels were voltage- and calcium-dependent. For comparison of the calcium sensitivity of different channels it is convenient to determine the concentration of calcium required to produce a half maximal opening at 0 mV. For cSlo1 that concentration falls between 10 and 20 µM (by interpolation). Previous measures for human brain Slo (hbr1) were 3 µM (DiChiara & Reinhardt 1995) and approximately 15 µM calcium (Dworetzky et al. 1996). Thus cSlo1 encodes a BK type potassium channel that is comparable to human brain Slo hbr1, with which it is highly homologous. Expression of cSlo in basilar papilla was demonstrated using RT-PCR on microdissected tissue. RTPCR also revealed that cSlo was expressed in tegmentum vasculosum, a secreting epithelium analogous to the stria vascularis of the mammalian cochlea. Thus, while cSlo is a strong candidate for the hair cell BK channel gene, it may be expressed in other cell types within the cochlea as well as in hair cells. Confirmation of cSlo expression by hair cells may be possible with more sensitive techniques such as single cell RT-PCR, or by the in situ polymerase chain reaction. Indeed, recent reports have indicated that this is the case (Navaratnam et al. 1997 ; Rosenblatt et al. 1997). How do cSlo1 channels compare with the native channels that carry BK currents in hair cells ? IK(Ca) carried by BK channels in chick hair cells is similarly sensitive to TEA and charybdotoxin (Fuchs & Evans 1990 ; Yuhas & Fuchs 1995), but comparable single channel data are not available. The single channel conductance of cSlo1 is like that of hair cell BK channels measured under similar conditions in frog (195 pS, Hudspeth & Lewis 1988), guinea-pig (233 pS, Ashmore & Meech 1986) and fish (130–320 pS, most commonly 250 pS, Sugihara 1994), but less than that found in turtle (320 pS, Art et al. 1995). The calcium sensitivity of BK channels varies with hair cell type. BK channels in electrically tuned hair cells of turtle appear to be most sensitive, with half maximal opening at 0 mV produced by approximately 5 µM calcium (Art et al. 1995). This is somewhat lower than the comparable value for cSlo1 expressed in HEK 293 cells (10–20 µM). Differences in calcium sensitivity between native turtle channels and cSlo1 become more pronounced at negative membrane potentials. At ®50 mV the turtle channels were half-activated by 12 µM calcium, while the half-activating calcium concentration for cSlo1 may be ten times higher (150 µM at ®50 mV, estimated from a plot of V" versus calcium– # not shown). The higher calcium sensitivity of turtle hair cell channels might arise from combination with auxiliary β subunits as shown for Slo channels from mammalian brain (McManus et al. 1995 ; Dworetzky et al. 1996). Therefore, it will be of interest to determine if Slo β subunits also are expressed in chick’s cochlea. This study demonstrates that a Slo gene is expressed in chicks’ basilar papilla and that it encodes channels similar to the native BK channels of hair cells. This is an important first step in the determination of the molecular basis of electrical tuning in hair cells, and inspires further study to determine the hair cell-specific expression of cSlo splice variants and related gene Proc. R. Soc. Lond. B (1997)

products. Furthermore, developmental regulation of BK channels in hair cells of the chick’s cochlea (Fuchs & Sokolowski 1990) can now be studied at the level of transcription of the cSlo gene. We thank Dr L. Salkoff for his gift of mSlo and Dr A. Ribera for advice and guidance throughout this work. The study was supported in part by research grant number 7 RO1 DC 00276 from the National Institute on Deafness and Other Communication Disorders (NIDCD) and by the National Organization for Hearing Research. R.L.M. was the recipient of a Reentry Supplement Award from NIDCD.

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