©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Permeation Properties and Differential Expression across the Auditory Receptor Epithelium of an Inward Rectifier K Channel Cloned from the Chick Inner Ear (*)

(Received for publication, March 31, 1995; and in revised form, June 5, 1995)

Dhasakumar S. Navaratnam (1) Laura Escobar (2) Manuel Covarrubias (2) J. Carl Oberholtzer (1)(§)

From the  (1)Department of Pathology and Laboratory Medicine, Division of Neuropathology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 and the (2)Department of Anatomy, Pathology and Cell Biology, Jefferson Medical College, Philadelphia, Pennsylvania 19107

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The auditory receptor epithelium is an excellent model system for studying the differential expression of ion channel genes. An inward rectifier potassium current is among those which have been measured in only subsets of chick cochlear hair cells. We have cloned and characterized an inward rectifier potassium channel (cIRK1) from the chick cochlear sensory epithelium. cIRK1 functional properties are similar to those of the native channel, and the transcript encoding cIRK1 is limited to the low frequency half of the epithelium. This localization is in agreement with the distribution of the native hair cell current, suggesting that the differential current expression is transcriptionally regulated. The primary structure of cIRK1 is highly homologous to the mouse inward rectifier IRK1. However, we found that cIRK1 exhibited reduced single-channel conductance (17 picosiemens) and lower sensitivity to Ba block (K = 12 µM). We identified Gln-125 near the putative pore region as being responsible for these differences. Site-directed mutagenesis was used to change Gln-125 to Glu (the residue in IRK1), resulting in a channel with a single-channel conductance of 28 picosiemens and a Ba block of K = 2 µM. We propose that Gln-125 may form part of the external vestibule of the pore.


INTRODUCTION

The auditory receptor epithelium of the chick, the basilar papilla, consists of hair cells and supporting cells. Approximately 10,000 hair cells are found in the receptor epithelium(1) . These cells receive either primarily efferent or primarily afferent innervation (2) . Those cells that receive predominantly afferent innervation (tall hair cells) are the primary receptors that transduce sound(3) . These cells convert the mechanical energy of sound to a coded electrochemical one(4, 5) . Tall hair cells are arranged tonotopically along the long axis of the basilar papilla with cells toward the apex responding to low frequency sound and cells toward the base responding to high frequency sound(6) . In the chicken, the membrane properties of tall hair cells differ along the tonotopic axis, and potassium channels, in particular, play a pivotal role in determining these properties(7, 8, 55) .

Electrophysiological experiments have shown three potassium currents in tall hair cells: a calcium-activated potassium current; a delayed rectifier potassium current; and an inward rectifier potassium current (8, 55) . Of these, both the inward rectifier and delayed rectifier currents have been shown to be present in hair cells responding to low frequency sound (apical hair cells), while the calcium-activated potassium current has been shown to be present in those hair cells responding to high frequency sound. The inward rectifier potassium current contributes to determining the resting membrane potentials and the membrane response times of these cells. The inward rectifier current present in tall hair cells has sharp inward rectification, prolonged open times, and is sensitive to block with external Ba and Cs(8) .

Several inward rectifier potassium channels with differing electrophysiological properties and mechanisms of activation have recently been cloned from a number of tissues and species. Among these are IRK1 from mouse macrophages(9) , ROMK1 from rat kidney(10) , GIRK1 (11, 12) and rcK(13) from rat heart, HIRK1 from human hippocampus(14) , and RB-IRK2 from rat brain and RBHIK1 from rabbit heart(15, 16) . Of these, GIRK1 is activated through a G protein, rcK is opened by ATP, and the remainder are activated by hyperpolarizing voltages. Apart from ROMK1, which has been shown to have moderate rectification, the rest of these channels have sharp inward rectification. All of these channels can be blocked by application of external cations (Ba and Cs). Hydropathy analyses of all these channels predict two membrane-spanning hydrophobic domains (M1 and M2) in the middle of the protein(9, 10, 11, 12) . These membrane-spanning domains are separated by the H5 region. The H5 domain forms an important part of the pore in potassium channels(17, 18) . The amino acid sequences of these inward rectifier channels are most conserved in the membrane-spanning and H5 regions. A number of experiments have helped elucidate their structure-function relations. Recently, it was reported that replacing the C terminus of ROMK1 with that of IRK1 altered its electrophysiological properties of Mg block and K conductance(19) . Furthermore, mutation of a single residue (Asp-172) in the M2 segment of IRK1 demonstrated that this residue is important in determining inward rectification through internal Mg block and the rate of activation(20, 21) .

We report here the cloning of an inward rectifier potassium channel (cIRK1) from the chick basilar papilla and demonstrate its localization in the apical half of the epithelium. cIRK1 has a reduced single-channel conductance and a decreased sensitivity to block with external Ba compared with the mouse inward rectifier, IRK1. Both differences were removed by substituting a glutamate residue for glutamine 125 in cIRK1. Glutamine 125 lies between the proposed M1 and H5 regions of the channel. This residue may be part of the external vestibule of the pore.


EXPERIMENTAL PROCEDURES

mRNA Isolation

Poly(A) RNA was isolated using oligo(dT)-cellulose (Micro-FastTrack, Invitrogen). For Northern blots, poly(A) RNA was isolated from 30 cochlea and from 200 mg each of brain, cerebellum, liver, heart, and muscle. Likewise, for reverse transcription-polymerase chain reaction (RT-PCR) (^1)experiments with the nested primers, poly(A) RNA was isolated from 15 microdissected basilar papillae (Micro-FastTrack, Invitrogen). For basilar papilla dissections, cochleas were first removed as described previously (22) and placed in cold Hanks'-buffered saline. Accessory tissues in the cochlear ducts were then dissected away from basilar papillae using the superior and inferior cartilaginous plates as visual and mechanical landmarks. In experiments in which RT-PCR experiments were done on cDNA from the apical and basal parts of the basilar papilla, 20 basilar papillae were microdissected as before, cut across their midpoints, and poly(A) RNA was isolated from each of the pooled halves as for the whole basilar papillae.

PCR

Four primers were designed based on conserved amino acid sequences between the two inward rectifiers, IRK1 and ROMK-1(9, 10) . The nucleotide sequences of these primers were AAAGAWGGNMGNTGXAAX (5`-3` KDGRCN), ACHACNADXTCRAARTC (5`-3` a`sense of DFEIVV), ACAATHGGNTAXGGN (5`-3` TIGYG), and CARRCANARXTTNCC (5`-3` a`sense of GKLCLL). The first two of the above primers were used in an initial amplification in a 100-µl reaction with the following parameters: 94 °C, 1 min; 45 °C, 2 min; 72 °C, 3 min; 40 cycles with 4 mM MgCl(2) and 0.5 unit of Taq polymerase (Perkin-Elmer Cetus). For these experiments, cDNA synthesized with random hexamers, avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim), and poly(A) RNA obtained from 15 microdissected basilar papillae served as the template. The amplified DNA contained in 10 µl of this initial reaction was then used as a template for a second round of amplification. This reaction was performed using the last two of the above primers where the conditions used were identical with those in the first round with the exception of annealing temperature, which was raised to 48 °C. The PCR products were subcloned into the TA cloning vector (Invitrogen) and sequenced (23) to determine their identity.

In experiments intended to identify the prevalence of these transcripts in the two halves of the basilar papilla, template cDNAs for PCR were synthesized by random priming of poly(A) RNA isolated from microdissected apical and basal halves of the basilar papilla. The primers used, based on the sequence of cIRK1, had the following sequences: TCTTCAGCCACAATGCCGTG and AGACCAGGAATATGCGGTC. Equal amounts of cDNA were used in each PCR. The amounts of cDNA were determined by colorimetry (DNA DipStick, Invitrogen). The PCR was carried out in a 20-µl volume with 4 mM MgCl(2), 5 µCi of [P]dCTP, and 0.5 unit of Taq polymerase using the following conditions: 94 °C, 1 min; 45 °C, 2 min, 72 °C, 3 min for 60 cycles. 2-µl aliquots were removed every 10 cycles from the 30th cycle onward. These aliquots were then separated on a denaturing polyacrylamide gel (6 M urea, 6% acrylamide, 1 TBE (90 mM Tris borate, 2 mM EDTA). The amount of radioactivity in the band of the anticipated size was determined using a PhosphorImager. Similar experiments were performed with primers specific to the chick calbindin-D28k sequence. The primers used had the following sequences: CAGGGTGTCAAAATGTGTGC and GGTCAAGACGAGCCATTTCG. They were based on the chick cDNA sequence (24) and were chosen to span several introns(25) . The experimental conditions were identical with those used in the experiment done to localize cIRK1, except that the annealing temperature was raised to 55 °C and the reaction was terminated after 30 cycles.

Library Screening

An oligo(dT)-primed ZAP chick cochlea cDNA library (26) containing approximately 2 10^6 independent recombinants was screened with the PCR products whose sequences most resembled those of IRK1 and ROMK-1. Random primed labeling (27) was used to generate the P-containing probe (Boehringer Mannheim). Hybridization was done in 6 SSC, 5 Denhardt's solution (50 is 1% Ficoll, 1% polyvinylpyrrolidone, 1% bovine serum albumin), 0.1% SDS at 65 °C for 16 h, and the filters were washed twice in 2 SSC at 65 °C and once in 0.1 SSC at 65 °C (1 SSC is 0.15 M NaCl, 15 mM sodium citrate). Six clones were identified with the inward rectifier probe. The plasmids were recovered by in vitro excision with the R408 helper phage (Stratagene), and the inserts were sequenced (23) with vector- and insert-derived primers. The final sequence was determined from both strands.

Northern Analysis

5 µg of poly(A) RNA from chick brain, cerebellum, muscle, liver, and heart and 1 µg of poly(A) RNA from chick cochlea were separated on a formaldehyde agarose gel(28) . Capillary transfer of the RNA to reinforced nitrocellulose was effected in 20 SSC, after brief alkali treatment(28) . The RNA was then fixed to the membrane by baking the membrane in a vacuum at 80 °C for 2 h. The Northern blot was probed with the entire cloned insert of the inward rectifier, which was random prime P-labeled(27) . Hybridization and wash conditions were as described above for the screening of the libraries.

Site-directed Mutagenesis

Mutagenesis was performed using the Altered Sites mutagenesis kit (Promega). Briefly, the entire insert was directionally subcloned into the pAlter-1 vector, single-stranded phagemid made with the aid of the helper phage R408, the mutant primer, and an ampicillin-resistant primer allowed to anneal to the phagemid and second strand DNA made with DNA polymerase and T4 DNA ligase. The double-stranded DNA was then transfected into JM109 (high efficiency) cells, and the presence of the mutation was confirmed by dideoxy sequencing. Those constructs with the mutation were then subcloned into a modified pBluescript vector for cRNA synthesis.

Electrophysiology

The wild-type and mutant cDNA were subcloned into pBluescript such that the 5` and 3` ends were flanked by the 5`- and 3`-untranslated sequence of the Xenopus beta-globin gene. This modified vector was made by Dr. Aguan Wei (Washington University, St Louis). Capped RNA was generated using T3 RNA polymerase with the linearized plasmid serving as a template (Ambion). Harvesting of oocytes and microinjection were done by standard procedures(29) .

Electrophysiological recordings from Xenopus oocytes were done using a TEV-200 two-microelectrode voltage clamp amplifier (Dagan), to record whole cell currents, or an Axopatch 200 patch-clamp amplifier (Axon Instruments), to record single-channel currents(30) . For whole-cell and cell-attached patch-clamp recording, the bath solution contained 140 mM potassium aspartate, 10 mM KCl, 1.8 mM CaCl(2), 10 mM HEPES (pH 7.4; titrated with KOH). In the cell-attached configuration, this solution clamps the membrane potential near 0 mV. The same solution was present in the patch pipette. Whole cell currents were low-pass-filtered at 1 kHz using an internal 4-pole Bessel filter and digitized at 1 or 2 kHz. Single-channel currents were low-pass-filtered at 1 or 2 kHz using an 8-pole Bessel filter and digitized at 4 or 8 kHz, respectively. Leak currents were subtracted assuming a linear leak at positive voltages, and capacitive currents in ramp experiments were subtracted using templates constructed by fitting a smooth function to records with no openings. BaCl(2) was applied using a gravity-driven perfusion system. To ensure equilibrium, the recording chamber was perfused with at least four chamber volumes (4 250 µl) at a rate of approximately 3 ml/min. All currents were recorded at room temperature (22-23 °C). Data acquisition and analysis were done using pCLAMP 6.0. (Axon Instruments). For further analysis we used Sigmaplot (Jandel). All results are reported as means ± S.D.


RESULTS

Cloning of an Inward Rectifier Potassium Channel (cIRK1) from the Chicken Basilar Papilla

Initial PCR analysis with degenerate primers using cDNA from microdissected basilar papillae revealed a 456-base pair product. The deduced amino acid sequence of this product most closely resembled that of the mouse inward rectifier IRK1(9) . We isolated six clones by screening an oligo(dT)-primed chick cochlea cDNA library with these PCR products. All six clones were of approximately the same size (2.2 kb) and had identical 3` ends. The 5` ends were similar and only differed in their degree of extension. The nucleic acid sequence of the longest of these clones is shown in Fig. 1together with the deduced amino acid sequence of its contained open reading frame (320-1601 nucleotides). Of note was the absence of a consensus polyadenylation signal before the 3`-poly(A) end. The 427-amino-acid protein, termed cIRK1, would have a molecular mass of 49 kDa. The nucleic acid sequence surrounding the initiator methionine matched the consensus Kozak sequence(31) . There was no signal peptide in the deduced amino acid sequence. The protein also contained the following other motifs for possible post-translational modification: four protein kinase A phosphorylation sites (amino acids 151, 192, 237, and 425), one N-glycosylation site (amino acid 190), one protein kinase C phosphorylation site (amino acid 6), and two tyrosine kinase phosphorylation sites (amino acids 242 and 336). The amino acid sequence was highly homologous to that of the mouse inward rectifier IRK1, with two exceptions: a stretch (amino acids 114-127) corresponding to the region between the purported M1 and H5 transmembrane domains and an additional group of amino acids (amino acids 388-413) near the C terminus ( Fig. 1and 2).


Figure 1: Nucleic acid and deduced amino acid sequences of cIRK1. The nucleic acid sequence of the longest inward rectifier clone and its deduced amino acid sequence are shown. The purported membrane-spanning and pore regions in the deduced amino acid sequence are underlined. The initiator methionine was ascribed according to the consensus Kozak sequence. Also indicated are the possible sites for post-translational modification (MacVector 4.1, IBI): an N-glycosylation site (#), one protein kinase C phosphorylation site (), four protein kinase A phosphorylation sites (+), and two tyrosine kinase phosphorylation sites (♦). Indicated below the deduced amino acid sequence are the amino acids which differed in IRK1 (9) . These are clustered around the region connecting the M1 and H5 regions and near the C terminus.



cIRK1 Is Present in the Apical Basilar Papilla

The size and distribution of the cIRK1 transcript(s) were determined by Northern analysis. This revealed an intense band of approximately 5.4 kb in size that was present in poly(A) RNA from cochlea, brain, muscle, cerebellum, heart, and liver (Fig. 3). A less intense band of 2.2 kb was also present in these tissues. Several other bands of lesser intensity and of varying sizes were also seen in these tissues; these persisted even after washing at high stringency.


Figure 3: Size and tissue distribution of cIRK1 transcripts. Northern analysis of poly(A) RNA obtained from brain, cerebellum, cochlea, muscle, liver, and heart. A DraI fragment (1.9 kb) of cIRK1 was used as a probe. The blot was washed in 0.1 SSC at 65 °C. The major band of 5.4 kb (arrow) with a less intense band of 2.2 kb and several minor bands of different sizes are present. The migration of the RNA size markers is indicated on the left (sizes in kilobases).



We then attempted to localize this channel within the basilar papilla. This was done by a PCR using primers specific to the cIRK1 clone. cDNAs obtained from the apical and basal halves of the basilar papilla were used as templates in two separate reactions. A PCR product of the anticipated size was seen only in the reaction which contained cDNA obtained from the apical half of the basilar papilla (Fig. 4). There was no evidence of such a product in the basal half of the basilar papilla even after 60 cycles. A parallel control experiment was done to confirm the integrity of the cDNA obtained from the two halves of the receptor epithelium. A similar PCR was performed using primers specific for calbindin D28k, a calcium binding protein abundant in the chick basilar papilla(32) . In this experiment, and in contrast to the result obtained with the cIRK1 primers, a product of the expected size was present in the PCR where cDNA from the basal half of the epithelium served as the template (Fig. 4). A much less intense product of the anticipated size was also present in the reaction in which cDNA from the apical half of the epithelium was used as the template (Fig. 4). Although we have not attempted to test the prevalence of these transcripts by competitive PCR, we are confident of these results since the difference in abundance of the calbindin transcript between the two halves of the basilar papilla was greater than two orders of magnitude (860,000 versus 3,000 units as measured by a PhosphorImager). Moreover, this ratio which was present at the 15th cycle of the PCR was maintained at the 30th cycle. Each reaction was done in triplicate with a view to controlling for differences in amplification efficiency. Furthermore, in order to control for possible variations in mRNA isolation and cDNA synthesis, these steps were repeated, and PCRs were performed on these samples with the same results (data not shown).


Figure 4: Differential distribution of cIRK1 transcripts along the tonotopic axis of the basilar papilla. A, the differential distribution of cIRK1 and calbindin transcripts across the basilar papilla (cartoon). The upper panel shows the products of RT-PCRs (in triplicate) with primers specific to cIRK1 after 30, 40, 50, and 60 cycles using cDNA obtained from the apical (left) and basal (right) halves of the basilar papilla. The product is seen only in the PCRs which had cDNA from the apical half of the basilar papilla. The lower panel shows the products from a similar experiment using primers specific to calbindin-D28k after 30 cycles. In contrast to the pattern obtained with cIRK1, the product is seen predominantly in the basal half of the epithelium. B, quantitation of PCR products from the two halves of the basilar papilla. The signals from the triplicate samples from each cycle point were measured as one value using a PhosphorImager. These values are plotted as a function of PCR cycle number for both the apical and basal halves of the basilar papilla.



The Electrophysiological Properties of cIRK1 Are Similar to Those Exhibited by the Inward Rectifier in Apical Hair Cells

The electrophysiological properties of cIRK1 were determined following expression in Xenopus oocytes. This was done by recording both whole-cell and single-channel currents. cIRK1 had electrophysiological properties similar to the inward potassium current in apical hair cells(8) . Whole-cell currents showed rapid activation and moderate inactivation over the length of the 900-ms pulse (Fig. 5A). cIRK1 also exhibited pronounced inward rectification. In the presence of high external potassium, little or no outward current could be detected at membrane potentials above the reversal potential of 0 mV (Fig. 5B). Single-channel currents also showed marked inward rectification (Fig. 5C and 6, A and C), and the single-channel conductance was 16.9 pS ± 1.7 (n = 4). The channels had a mean open time of 210 ms at -150 mV (n = 2).


Figure 5: Electrophysiological properties of cIRK1 expressed in Xenopus oocytes. A, whole-oocyte currents evoked by 900-ms step voltage changes between -150 and +50 mV in 20-mV increments from a holding voltage of 0 mV. Currents were elicited at intervals of 5 s. Data were low-pass filtered and digitized at 1 kHz. Leak current was subtracted assuming that a linear component of the current-voltage relation at positive voltages was mainly due to leak. This seems a reasonable assumption because unsubtracted macroscopic recordings from tight-seal macropatches showed little or no current at positive potentials, and single-channel recording showed no openings above the expected reversal potential (-10 to 0 mV). Capacitive transients were not subtracted. B, the corresponding steady-state current-voltage relation. Current was measured as the average of at least 10 sampling points near the end of the pulse. C, single-channel currents from a cell-attached patch recorded at the indicated membrane voltages. Data were low-pass filtered at 1 kHz and digitized at 4 kHz. D, corresponding single-channel current-voltage relation. Each point represents the mean current amplitude of single-channel openings. Solid line is the best-fit linear regression with a slope of 16 pS. For all recordings the external solution contained 150 mM K (see ``Experimental Procedures'').



A Single Glutamine Residue Influences Conductance and Block by External Ba

The overall electrophysiological properties of cIRK1 were similar to IRK1. However, the single-channel conductance of cIRK1 was found to be 17 pS which compares with a value of 23 pS found in IRK1(9) . A single amino acid (Gln-125) was identified as possibly being responsible for the reduced conductance. We chose this residue because it was different in the two channels, was adjacent to the H5 region, and since its change from a negatively charged residue (glutamate in IRK1) to a polar noncharged residue (glutamine in cIRK1) could account for the reduced conductance (Fig. 2). Accordingly, this amino acid was changed to a glutamate residue by site-directed mutagenesis. The mutated channel (Q125E) was expressed in oocytes, and its conductance was determined from steady-state single-channel recordings and ramp currents. As shown in Fig. 6A, changing the glutamine residue to a glutamate resulted in a channel with a single-channel conductance of 28.3 pS ± 3.9 (n = 4). Thus, Q125E had an increased conductance as compared to wild-type cIRK1; a conductance which would be predicted to be similar to that of IRK1 when measured under identical conditions (our measurements were at 150 mM K; 140 mM K was used in (9) ). There was no significant effect of the mutation on the kinetic properties or inward rectification (Fig. 7).


Figure 2: Alignment of cIRK1 with representative members of the inward rectifier family in the region between M1 and H5. The Clustal method (54) was used to align the amino acid sequences. Amino acid identities with cIRK1 are shown as dashes; gaps in sequences introduced for alignment purposes are shown as blank spaces. Position 125 in cIRK1 (Q) and IRK1 (E) and corresponding positions in other sequences are shown in bold. The overall amino acid similarity between cIRK1 and the different channels are: IRK1, 93% (9) ; RB-IRK2, 69%(15) ; RBHIK1, 94%(16) ; HRK1, 58%(14) ; GIRK1/KGA, 44%(11, 12) .




Figure 6: The effect of Q125E on single-channel conductance of cIRK1. A, single-channel ramp current expressed by wild-type cIRK1. The trace shown is the average of five consecutive responses. Voltage ramp was delivered at 2.2 mV/ms. Data were filtered at 1 kHz and digitized at 20 kHz. Solid straight line represents the best-fit linear regression with a slope of 19 pS (calculated between -150 and -10 mV). B, single-channel ramp current expressed by mutant cIRK1 (Q125E). Voltage ramp was delivered at 0.44 mV/ms. Current is interrupted by a brief complete closure of the channel. Data were filtered at 1 kHz and digitized at 4 kHz. The estimated slope conductance was 33 pS (solid straight line). C, single-channel current-voltage relation determined from steady-state records from oocytes expressing wild-type (n = 3) and Q125E (n = 1). Mean current amplitude at each voltage was determined from amplitude histograms. Standard deviation bars are not apparent when they are not larger than the symbol. Slope conductances (solid lines) were 16.3 and 25.6 pS for wild-type and mutant, respectively. Estimated pooled averages from steady-state and ramp experiments were 16.9 ± 1.7 (n = 4) and 28.3 ± 3.9 (n = 4), wild-type and mutant, respectively. All recordings from cell-attached patches with 150 mM K in the recording pipette.




Figure 7: The effect of Q125E on the sensitivity of cIRK1 to Ba block. A and C, whole-oocyte cIRK1 currents in the absence (control) and presence of 5 µM BaCl(2) from oocytes expressing wild-type and mutant channels, respectively. Currents were recorded and analyzed as indicated in the legend to Fig. 5. B and D, isochronal (end of 900-ms pulse) current-voltage relations in the presence of increasing external concentrations of BaCl(2) for wild-type and mutant cIRK1. E, dose-response experiment at -150 mV. Points and bars represent the mean ± S.D. from 5 (wild-type) and 3 (Q125E) oocytes. Solid line represents the best least-squares fit to: y = 1/(1 + (x/K)), where K is the value of x causing 50% inhibition. The best-fit parameters were K = 11.73, n = 1.4 for wild-type; and K = 2.1, n = 1.6 for Q125E. Dotted lines are curves drawn assuming the same values for K but n = 1.0.



We also studied the effect of Ba on cIRK1. It was observed that cIRK1 currents appeared to be somewhat less sensitive to block with Ba than did those of the mouse inward rectifier. For instance, IRK1 was completely blocked by 30 µM Ba by 900 ms at both -130 and -160 mV(9) , while the same concentration of Ba resulted in only a 70% average reduction in cIRK1 at -150 mV at 900 ms (the end of our pulse, Fig. 7). The ability of Ba to block current in Q125E was also determined. We had observed that the sensitivity of cIRK1 to Ba block was to some degree inversely proportional to the level of expression; this effect was especially apparent for currents > 10 µA. (^2)Thus, to study the effect of Ba on wild-type and mutant channels, we studied oocytes expressing currents within a range of 1-10 µA (at -150 mV). Compared to the wild-type channel, Q125E had an increased sensitivity to block with external Ba (Fig. 7). The best-fit parameters of a Langmuir isotherm were: K (WT) = 12 µM, n(H) (WT) = 1.4; and K (Q125E) = 2 µM, n(H) (Q125E) = 1.6. Although the apparent binding affinity K was increased about 6-fold, the Hill coefficients (n(H)) were similar.


DISCUSSION

Electrophysiological experiments have demonstrated an inward rectifier potassium current to be present in apical hair cells(8, 55) . It plays an important role in determining the excitable properties of these cells, including the resting membrane potential and the membrane time constant. We have cloned an inward rectifier potassium channel from the chicken basilar papilla. We have termed this channel cIRK1 for its close homology to the mouse inward rectifier (IRK1). Two arguments would favor cIRK1 being the same as the inward rectifier potassium channel present in tall hair cells. First, the electrophysiological properties of cIRK1 are similar to those of the inward rectifier channel present in apical hair cells. In particular is its pronounced inward rectification, which is an important feature of the inward rectifier channel in apical hair cells. Second, we were able to demonstrate by RT-PCR that transcripts encoding the channel were present in the apical half of the basilar papilla, a result which parallels the electrophysiological data. Although we obtained a PCR product whose sequence resembled the inward rectifier ROMK-1, it is unlikely that this channel would be responsible for the inward rectification in apical hair cells. This is because of an inability to obtain a clone bearing this sequence on screening cDNA libraries of 2 10^6 recombinants, and because ROMK-1 displays poor inward rectification(10) .

The limitation of the cIRK1 transcript to the apical half of the basilar papilla and the distribution of the calbindin transcript predominantly in the basal half of the basilar papilla would suggest that the differential distribution of these proteins is determined by control of transcription. This is direct evidence for such control within the inner ear epithelium. Should this process extend to other channels, as is suggested by the electrophysiological data, it would imply that a complex transcriptional regulatory process contributes to bringing about the tonotopicity in the basilar papilla.

The preferential distribution of the calbindin transcript in the basal half of the receptor epithelium also has implications to electrical tuning. Electrical tuning is a mechanism by which the frequency of oscillation in membrane potential defines the frequency of sound to which a given hair cell best responds. It was first demonstrated in the turtle(33) , and there is evidence that it also occurs in the chick (8) , although some have questioned its significance in the latter(34) . The graded oscillations in membrane potential of hair cells across the epithelium are brought about by an interplay between calcium-activated K channels and calcium channels in individual hair cells (35) . The frequency of oscillation in membrane potential of a given hair cell is determined by the individual properties of the calcium-activated K channel peculiar to that particular hair cell or by the calcium buffering properties particular to that cell(36) . It has been shown that the concentration of a cytoplasmic calcium buffer within hair cells is sufficiently high to cause a spatial buffering of Ca within hair cells(37) . Calbindin is thought to serve such a function(32, 37, 38) . The differential distribution of the calbindin transcript within the sensory epithelium would suggest that an incremental Ca buffering by calbindin could contribute to determining the graded oscillation frequencies and electrical tuning.

The six clones of 2.2 kb obtained on screening the cDNA library are unlikely to be full-length clones, even though they contained the entire open reading frame. This is suggested by the absence of a polyadenylation signal before the 3`-poly(A) end, and the major transcript of 5.4 kb present on Northern analysis of the different tissues including the cochlea. Furthermore, the transcripts encoding the analogous channels in the mouse (9) and the rat (16) are 5.4 kb in size. Since all the clones were of 2.2 kb in size, it would necessitate that the oligo(dT) primer primed at a site, rich in adenosines, internal to the poly(A) tail when the cDNA libraries were constructed. However, we cannot rule out the possibility that the cloned cDNA was derived from the 2.2-kb transcript detected on Northern analysis. The several less intense bands detected on Northern analysis in all these tissues would imply the existence of several other related mRNAs (channels) in these tissues.

All six clones obtained from the cDNA library had the same sequence and were not alternatively spliced. Furthermore, the gene for IRK1 in the mouse has been shown to be intronless within the open reading frame (39) . That there were no differences in the electrophysiological properties of the inward rectifier potassium currents in hair cells obtained from different locations in the apical basilar papilla is in keeping with these data(8) .

The differences in the deduced amino acid sequence of cIRK1 compared to the mouse channel (IRK1) were limited to two regions of the protein and may have several implications. One region of difference was the short sequence connecting the purported M1 and H5 regions. We identified a single, polar but uncharged amino acid (Gln-125) lying within this region of cIRK1 as being of importance in determining single-channel conductance and sensitivity to Ba block. The corresponding position in IRK1 contains a glutamate residue (which at physiological pH would have a negative charge). cIRK1 had a reduced single-channel conductance (17 pS using 150 mM external K) and reduced sensitivity to block with Ba compared to IRK1. IRK1 had a single-channel conductance of 23 pS when using 140 mM external K(9) . Consistent with this observation, a human homologue of IRK1, HIRK1(14) , which has a histidine in this position, was found to have a single-channel conductance of 10 pS as measured under somewhat different experimental conditions (100 mM external K). IRK1 has been shown to have a single-channel conductance of 21 pS when using 100 mM external K(19) . Changing glutamine 125 in cIRK1 to a glutamate residue resulted in a channel which had a single-channel conductance of 28 pS (using 150 mM external K). It also had an increased sensitivity to Ba block. The K value for Ba block in the wild-type channel was 12 µM, while that for Q125E was 2 µM. Although other inward rectifier channels have been cloned that have different amino acids in this position, it is difficult to compare these owing to the absence of directly comparable electrophysiological data. Of particular interest are the rat brain inward rectifier RB-IRK-2 (16) and the rabbit cardiac channel RBHIK1 (15) which contain a glutamine and glutamate, respectively, in this position.

The altered single-channel conductance and sensitivity to Ba block in cIRK1 that was brought about by changing Gln-125 to a glutamate lends itself to two explanations. First, it is possible that Gln-125 lines the pore or the vestibule of the channel. Second, it is also possible that the change in the electrophysiological properties of the channel produced by this mutation are mediated by an allosteric effect. Since two independent properties of the channel were changed in a manner consistent with a direct interaction (see below), the first of these possibilities is the more likely. This interpretation is also consistent with the proposed membrane topology of the channel. Both Gln-125 in cIRK1 and Glu-125 in IRK1 are preceded by a series of charged amino acids (amino acids 112-117). A fortuitous change in one of these amino acids between these two channels enables us to make a prediction about its contribution to conductance and sensitivity to Ba block. A single glutamate residue (Glu-117) in cIRK1 occupies the position of a lysine in IRK1. Such a reversal in polarity would be predicted to increase the conductance and sensitivity to external cation block in cIRK1 (see below). That it does not would argue that this amino acid (Glu-117) does not contribute to determining either of these properties.

The calculated Langmuir isotherms permit us to deduce the importance of Gln-125 and by inference Glu-125 in IRK1, to Ba binding. Consistent with a mechanism involving multi-ion block(40, 41, 42, 43) , we found a Hill coefficient > 1. This would suggest that Ba interacts at two (or more) sites. The mutant channel (Q125E) had the same Hill coefficient, albeit with an increased sensitivity. Although Gln-125 may form part of the Ba binding site within the pore, our results are also consistent with a hypothesis that Glu-125 in IRK1 may lie in the vestibule of the pore and influence conductance and Ba block by controlling diffusion of ions therein(44) . This is analogous to changes in conductance mediated by negatively charged residues at the outer vestibule of the nicotinic acetylcholine receptor channels(45) .

While Asp-172 in IRK1 is important in determining inward rectification mediated by internal Mg block(20, 21) , our results suggest that Glu-125 is an external residue that may play an important role in controlling channel conductance. Both actions may involve electrostatic interactions(46) . If K channels are tetrameric and exhibit 4-fold symmetry(47, 48) , Asp-172 and Glu-125 may form negatively charged rings at the inner and outer mouths of the pore, respectively.

cIRK1 differed from IRK1 near the C terminus (amino acids 388-413). The C terminus has been shown to confer some of the electrophysiological properties to the inward rectifiers. Specifically, replacing the C terminus of ROMK-1 with that of IRK1 resulted in the chimeric channel having rectification properties of IRK1 and a conductance intermediate between that of IRK1 and ROMK-1(19) . The electrophysiological properties of cIRK1 were, with the exception of conductance and sensitivity to external Ba block, not measurably different from those published properties of the mouse inward rectifier (IRK1). These two properties that were different were shown to be mainly due to a single amino acid change (Gln-125). Therefore, it could be reasoned that the amino acids that are different at the C terminus are not critical to determining these functional differences between the two channels. Since rectification of the two channels was the same, an extension of the same argument would suggest that these C-terminal amino acids that are different are not critical, or not sufficiently different, to affect this channel property.

At least three different potassium channels have been shown in electrophysiological experiments to be localized to different parts of the chick basilar papilla. Our results demonstrating the existence of cIRK1 transcripts within the apical basilar papilla would suggest that this channel, and, by implication, the other potassium channels within the basilar papilla are transcriptionally regulated. This would in turn necessitate complicated and minutely precise transcriptional regulatory mechanisms to bring about the differential expression of these and other proteins that determine tonotopicity within the 10,000 hair cells in the basilar papilla. Furthermore, our results relating to the alteration in conductance that is brought about by mutating Gln-125 has implications owing to a number of inward rectifier channels that differ in their conductances(49, 50, 51, 52) . This result suggests that one possible mechanism of altering conductance would be through changes in the external pore region. We would predict that this would be a site for natural mutations to occur thereby controlling conductance as physiologically required. This is particularly so since alteration of this residue does not seem to affect other channel properties such as its rate of activation and rectification.


FOOTNOTES

*
This work was supported by NIDCD Grant K08-DC-00069 and Pennsylvania Lions Hearing Research Foundation Grant GA-1307 (to J. C. O.), by NINDS Grant NS32337-02 (to M. C.), by grants from the Hospital de Especialidades, IMSS, Mexico (to L. E.), and by the Lucille P. Markey Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This paper is dedicated to J. D. Priddle.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) U20216[GenBank].

§
To whom correspondence and reprint requests should be addressed: Rm. 264 John Morgan Bldg., Dept. of Pathology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, Philadelphia, PA 19104. Tel.: 215-573-3272; Fax: 215-573-7738.

(^1)
The abbreviations used are: RT-PCR, reverse transcription-polymerase chain reaction; pS, picosiemens; kb, kilobase(s).

(^2)
This effect was not studied in detail, but was evident in the primary data from several experiments at different current levels. Given the strong voltage dependence of the barium block, one possible technical explanation for such a result is that the voltage control was inadequate. We do not believe this was the case in our studies since the kinetics and voltage dependence of our macropatch currents were similar to our whole oocyte currents over the 2-15 µA range. In addition, a similar result was reported recently (53) in which a plant-inward rectifier potassium channel was found to be less sensitive to cesium block when expressed at high levels in oocytes. The effect was studied in some detail by those investigators, including through the use of a third intracellular microelectrode, and appears to be physiological rather than technically artifactual. The mechanisms responsible for this phenomenon, and its biological consequences, remain obscure.


ACKNOWLEDGEMENTS

We would like to thank Dr. Mark Greene for extending the facilities in his laboratory and Dr. Jackie Tanaka for early experiments relating to expression. We would also like to acknowledge our indebtedness to Drs. Dick Horn, Jim Eberwine, and Jackie Tanaka for helpful comments on the manuscript.


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