1 Departments of Otolaryngology and Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston Texas, 77555-1063
2 Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston Texas, 77555-1063
3 Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston Texas, 77555-1063
4 Department of Otolaryngology, University of Texas Medical Branch, Galveston Texas, 77555-1063
5 Departments of Otolaryngology and Physiology and Biophysics, University of Colorado Health Sciences Center, Denver Colorado
6 Sealy Center For Environmental Health and Medicine, University of Texas Medical Branch, Galveston Texas, 77555-1063
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ABSTRACT |
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pigeon; potassium channel
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INTRODUCTION |
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Inwardly rectifying conductances have been measured in sensory epithelial cells (hair cells) of the inner ear of several species. These conductances have been studied in the developing and the mature cochlea (1517, 37, 38, 48, 62) and in the vestibular labyrinth (4, 23, 3942, 47, 58, 65).
In 1993, Ho et al. (21) and Kubo et al. (32) first reported the cloning, by expression, of an inward rectifier channel. Since then, using expression cloning and homology screening, a novel family of inward rectifier potassium channel genes (Kir) has been studied revealing seven subfamilies designated, using the nomenclature of Chandy and Gutman (5) and Gutman et al. (19), as Kir1.0 through Kir7.0.
Previously, only one study (45) has cloned an inward rectifier found in cochlea or vestibular hair cells. In that study, a gene named cIRK1 was isolated from sections of different regions of the auditory papilla (including hair cells, supporting cells, and nerve terminals) of the chick which was subsequently expressed in oocytes. Single-channel and whole cell patch clamp recordings from oocytes revealed a current with a single-channel conductance of 16 pS and a barium block sensitivity of EC50 = 12 µM. This single-channel conductance is only 70% (32, 34), and the EC50 is roughly five times that determined previously for other cloned Kir2.1 channels carrying IKir currents (61, 67).
Herein, we describe a series of experiments in which we isolated for the first time a gene (pKir2.1) coding for an inward rectifier channel in single vestibular hair cells of the pigeon. We present evidence from electrophysiological, pharmacological, immunohistochemical, and biochemical studies suggesting that the gene encodes a Kir2.1 potassium ion channel.
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MATERIALS AND METHODS |
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RT-PCR Cloning of pKir2.1
Total RNA was isolated from pigeon vestibular semicircular canal ampullae (including hair cells, supporting cells, and nerve terminals) using guanidinium thiocyanate (7). First-strand cDNA was synthesized in a final volume of 20 µl containing 20 mM Trisu·uHCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl2, 10 mM dithiothreitol, 500 µM of each dNTP, 0.5 µg oligo(dT)1218, and 200 U of SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). PCR amplification was performed using a 100-µl reaction mixture containing 20 mM Trisu·uHCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 µM of each dNTP, 110 µl cDNA, 5 U of AmpliTaq (PerkinElmer, Wellesley, MA), and 100 pmol of each primer. Each reaction was denatured for 2 min at 95°C prior to 3035 cycles at 94°C for 15 s, 60°C for 30 s, and 72°C for 4 min. The final primer extension was continued for 10 min at 72°C to enhance complete synthesis of the amplified target DNA. The initial PCR amplifications were performed using primers C1C4 listed below in Table 1, which were designed based upon the nucleotide sequence of the potassium inward rectifier channel, cIRK1, cloned from the chicken basilar papilla (45).
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Single cell RT-PCR
Because it was known that pKir2.1 was in hair cells and surrounding supporting cells (39) but unknown whether pKir2.1 was expressed in abutting vestibular nerve terminals (calyxes and boutons) and/or unmyelinated nerve fibers, all of which can be found in excised vestibular end-organs (ampullae and maculae), single type I and type II hair cells in slices through the pigeon ampullary and utricular epithelium (39, 65) were identified and whole cell patch clamped. Inwardly rectifying whole cell IKir ionic currents were obtained from type II hair cells (see Figs. 1C and 3A); the cytoplasm was aspirated (Fig. 3B, ac), and single-cell quantitative RT-PCR was carried out using primers C3 and C4 (see Fig. 3C).
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First-strand cDNA synthesis was performed using SuperScript II (Invitrogen) and conditions recommended by the manufacturer. PCR amplification was performed in a 100-µl reaction containing 20 mM Trisu·uHCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 200 µM of each dNTP, 5 U of AmpliTaq (PerkinElmer), and 100 pmol of each primer (5' AGACATCAATGTAGGGTTTGACAGC 3' and 5' AGATGGGTGTGTTGGGCACTTCG 3'). These primers were specific for the pKir2.1 ORF sequence. End-labeled ([-32P]ATP) primers (46 x 106 cpm/pmol) were added (13 x 106 cpm) for both primers to the PCR reaction. Following a denaturation step at 95°C, for 2 min, reactions were incubated for 35 cycles of 94°C for 15 s, 62°C for 30 s, and 72°C for 2 min. The final primer extension was continued for 10 min at 72°C. An internal standard RNA representing a 59-base deletion in the pKir2.1 target sequence (
pKir2.1) was added (100 molecules) to each RT-PCR assay. This reference RNA was used to quantify the pKir2.1 transcripts in each sample (2). A 20-µl sample from each reaction was electrophoresed through a 5% polyacrylamide gel (Fig. 3D) and analyzed using a PhosphorImager (Molecular Devices, Sunnyvale, CA).
Multiple Tissue RT-PCR
Total RNA was isolated from pigeon ampullae, maculae, vestibular ganglia, lens, neck muscle, brain stem, cerebellum, optic tectum, liver, and heart using the ToTALLY RNA kit (Ambion, Austin, TX). First-strand cDNA synthesis was performed using the RETROscript kit (Ambion) following the manufacturers protocol. PCR amplification was performed using the same primers (specific for pKir2.1 ORF) as were used for single cell qRT-PCR. Also, primers specific to the L19 ribosomal protein gene, were used as a positive control (sense 5'-TGAGGAGGATGCGGATCCTGAGG-3', antisense 5'-TGCCTTCAGCTTGTGGATGTGCTC-3'). The reaction mixture contained 10 mM Trisu·uHCl pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin, 200 µM of each dNTP, 5 µl cDNA, 50 pmol of each primer, and 5 U AmpliTaq DNA polymerase (PerkinElmer) in a total volume of 100 µl. The cycle conditions for the PCR were as follows: denaturation at 94°C for 30 s, primer annealing at 60°C for 30 s and primer extension at 72°C for 2 min for a total of 40 cycles. The final primer extension was expanded to 10 min to allow for full extension of incomplete products. Amplification products were analyzed by electrophoresis using 5% nondenaturing polyacrylamide gels and visualized by ethidium bromide (1%) staining.
pKir2.1 Expression in Oocytes
Oocyte vector.
To develop a functional assay for pKir2.1, we cloned the pKir2.1 ORF into pOCYT7, a vector modified from pTLN (35), to contain a multiple restriction site polylinker. The pTLN vector contains the Xenopus ß-globin 5' and 3' untranslated regions that have been reported to enhance expression of exogenous mRNAs following injection into Xenopus oocytes (31). The respective 5' and 3' ß-globin untranslated sequences flank the polylinker sequence in pOCYT7. In vitro transcription was performed with a linearized plasmid DNA NdeI and the mMessage mMachine (Ambion) using conditions recommended by the manufacturer. Following DNase I treatment (100 U/ml; 37°C, 15 min), Proteinase K (Roche/Boehringer Mannheim, Indianapolis, IN) was added to a final concentration of 500 µg/ml, and the reaction was incubated at 37°C for 1 h. RNA was isolated by either oligo(dT)-cellulose chromatography (1) or extraction with an equal volume of phenol-chloroform (50:50 vol/vol). In either case, RNA was collected by ethanol precipitation and dissolved in 10 mM Trisu·uHCl, pH 7.5.
Oocyte preparation and cRNA injection.
Surgery was performed on anesthetized (MS-222) female African clawed frogs (Xenopus laevis). Oocytes were removed and dissociated (overnight) at 17°C using collagenase (1 mg/ml) in Barth solution. The Barth solution contained (in mM) 1 KCl, 88 NaCl, 2.4 NaHCO3, 5.0 Trisu·uHCl, 0.82 MgSO4, 0.33 Ca(NO3)2, and 0.41 CaCl2. The oocytes were then washed several times in a calcium-free solution containing (in mM) 2.0 KCl, 82.5 NaHCO3, 1.0 MgCl2, and 5.0 HEPES followed by several washes in Barth solution and then stored for 34 h at 17°C. Fifty nanograms of cRNA was microinjected (Drummond Scientific, Broomall, PA) into stage II oocytes following the methods of Mo and colleagues (43). The injected oocytes were stored in Barth solution, and every day the solution was changed and dead oocytes were removed.
Oocyte electrophysiology.
At 2448 h postinjection, ionic currents were recorded from the oocytes using two-electrode voltage clamp methods. The recording pipettes were filled with 3 M KCl and had impedances of 1 M
. The bath was ND96 containing (in mM) 2 KCl, 98.0 NaCl, 1.0 MgCl2, 1.8 CaCl2, and 5.0 HEPES plus amikacin (100 µg/ml) and streptomycin (100 µg/ml). The amplifier that was used was a Heka EPC-7 (Instrutech, Port Washington, NY).
While the pKir2.1 ionic currents and their response to blockers in oocytes were qualitatively similar to those recorded in native hair cells, the time course of the inactivation of the pKir2.1 currents in oocytes was an order of magnitude longer than the IKir currents in native hair cells. To obtain a heterologous expression system (HES) with physical dimensions more like hair cells, we developed techniques to transfect pKir2.1 into mammalian Chinese hamster ovary (CHO) cells.
CHO Cell Culture and Plasmid Transfection
CHO-S cells were obtained from the Recombinant DNA Laboratory at UTMB and maintained but split once a week in complete media in T75 flasks in an incubator at 37°C, 95% relative humidity and 5% CO2. The complete media contained Dulbeccos modified Eagles medium (DMEM; Invitrogen/GIBCO, Carlsbad, CA), 10% fetal bovine serum (heat-inactivated, Invitrogen/GIBCO), 1x nonessential amino acids (Sigma, St. Louis, MO), and 1x penicillin/streptomycin/neomycin (Invitrogen/GIBCO). Following trypsinization, CHO cells were split from T75 flasks, counted with a hemocytometer, and plated into 35-mm petri dishes at a density of 3 x 105 cells/dish (2 ml). The cells were maintained up to 72 h in 2 ml of complete media. After 25 passages the cells were discarded, and new cells were obtained.
When the cells were 4080% confluent, they were transfected, using liposome-mediated methods, with plasmids encoding pKir2.1 ORF and enhanced green fluorescent protein (EGFP). In some experiments, pKir2.1 and EGFP were cloned into separate vectors (pcDNA3 and pEGFP-N1; BD Biosciences/Clontech, San Jose, CA). These plasmids were cotransfected into CHO cells. In other transfection experiments, both pKir2.1 and EGFP were expressed in the same vector (pCMS-EGFP, BD Biosciences/Clontech), or pKir2.1 was cloned in-frame with the COOH terminus of EGFP (pEGFP-C1, BD Biosciences/Clontech) or the NH2 terminus of EGFP (pEGFP-N1, BD Biosciences/Clontech).
Lipofectamine 2000 (Invitrogen/GIBCO) and Polyfect (Qiagen, Valencia, CA) were used as transfectants. In the former case, 1012 µg of DNA was used per 10 µl of Lipofectamine 2000; in the latter case, 1.53.0 µg of DNA/10 µl of Polyfect was used. The remaining procedures were according to the manufacturers instructions. The transfection efficiency was 6080%. The cells were incubated posttransfection for 24, 48, and 72 h. The cells were removed from the 35-mm petri dishes using a nonenzymatic dissociation solution (Specialty Media, Phillipsburg, NJ) and centrifuged at 500 g. After the supernatant was removed, the cells were resuspended in the extracellular bath solution and plated on the coverslip bottom of the recording dish, which had previously been coated twice with poly-D-lysine (0.5 µg/ml). In other cases, cells were cultured on 25-mm circular coverslips. Surface tension held the coverslips to the recording dish bottom.
CHO Cell Electrophysiology
Electrodes, pulled on a P-2000 puller (Sutter Instruments, Novato, CA), were made from blanks with an internal filament. The glass was either quartz (0.75 ID, 1.50 OD; Sutter Instruments) or borosilicate (0.75 ID, 1.50 OD, no. 1B150F-3; World Precision Instruments, Sarasota, FL,). To prohibit mRNA from adhering to the walls of the pipettes during cytoplasm extraction, the borosilicate or quartz blanks were initially washed with Chromerge (Fisher Scientific, Hampton, NH), silanized with 5% dimethyldichlorosilane (Sigma) in chloroform (Electron Microscopy Sciences, Hatfield, PA), then subsequently washed multiple times and dry heat sterilized at 240°C. The whole cell recording electrode impedance was typically 25 M. The single-channel electrode impedance was typically 25 M
, but often to isolate a single channel, electrodes were used with impedances as high as 30 M
. Series resistance and capacitance were compensated between 80 and 98% with a typical value of 95%. No online leak subtraction was performed. Subsequently, in some cases, leak subtraction was performed digitally. Whole cell currents were amplified 1x or 0.5x, and single-channel currents were amplified 100200x, using an Axopatch 200 amplifier (Axon Instruments, Union City, CA). Signals were acquired using a Digidata 1200 interface (Axon Instruments) or a 16-bit analog-to-digital interface (National Instruments, Austin, TX). The whole cell and single-channel currents were filtered at 2 kHz and sampled at 5 kHz. For the single-channel recordings, the system noise was 0.5 pA.
Stimulus waveforms for single-channel cell-attached on-cell recordings included voltage ramps and pulses over the range from 40 to 180 mV ( = 20 mV). Positive shifts in applied voltage were hyperpolarizing, and positive current was inward. For whole cell recordings the same applied voltages were used but with inverted sign and the current polarities were opposite. Current clamp whole cell recordings were made using the Axoclamp 2A amplifier (Axon Instruments), and pulse stimulation ranged from 20 to 100 nA (
= 20 nA).
Cells that showed green fluorescence, when viewed using FITC epifluorescence and differential-interference contrast (DIC) transmitted light, were selected for patch clamp recording. The cells were viewed through the optics of an Axophot microscope (Carl Zeiss, Oberkochen, Germany). During single-channel on-cell patch clamp and during whole cell recording, the bath consisted of (in mM) 130.0 KMeSO4, 20.0 KCl, 2.0 CaCl2, 5.0 HEPES, and 5.0 glucose. The pipette solution contained the same substances as the bath. In some experiments, the bath (extracellular) and pipette solutions used were the same as used for native cells in the labyrinthine slice (see below).
Native hair cell Electrophysiology
Hair cells in labyrinthine slices were patch clamped in whole cell mode using methods previously described (39, 65). Briefly, ampullae and utricles were harvested and dissected free of each other. The tissue was then incubated in DMEM augmented with 24 mM NaHCO3, 15 mM PIPES, 50 mg/l ascorbate, and 1.5% fetal calf serum. Tissue and medium were maintained at 37°C, pH 7.4, and an osmolality of 320 mosmol/kgH2O, in a saturated 95% O2-5% CO2 environment. At varying intervals, an ampulla or a utricle was removed from the incubator, embedded in 4% agar, and quickly covered by partially frozen Vibratome bath solution containing (in mM) 3.0 KCl, 145.0 NaCl, 0.1 CaCl2, 7.5 MgCl2, 15 HEPES, 10 glucose, 50 mg/l ascorbate, and 2 sodium pyruvate. The tissue was sliced, using a vibratory microtome (Campden, London, UK). Individual slices (150250 µm in thickness) were then transferred to a dish with a no. 1 glass coverslip bottom, held in place by a weighted nylon mesh and bathed in an oxygen saturated bath solution containing (in mM) 3.0 KCl, 145.0 NaCl, 2.0 CaCl2, 1.0 MgSO4, 15 HEPES, 10 glucose, 50 mg/l ascorbate, and 2 sodium pyruvate. The bath solution was titrated to a pH of 7.4 with NaOH and HCl and to an osmolality of 320 mosmol/kgH2O. The dish was mounted on a microscope stage (Zeiss Axioskop), and the cells were viewed using DIC microscopy optics including an Optovar magnifier and a 40x water-immersion objective. The temperature of the bath was 23°C and changed at a rate of 1.2 ml/min. The patch pipettes contained (in mM) 140.0 KCl, 1.0 CaCl2, 2.0 MgCl2, 10 HEPES, and 11 EGTA. The pipette solution was titrated to a pH of 7.4 with KOH/HCl and an osmolality of 315 mosmol/kgH2O.
Native Hair Cell and CHO Cell Pharmacology
Channel blockers were superfused onto the cell using a pressurized computerized superfusion system (model DAD-12; ALA Scientific Instruments, Westbury, NY). The superfusion pipette (100 µm bore) was placed 30 µm from the cell, and the flow rate varied from 5 to 1 µl/s.
Immunocytochemistry
Pigeons were perfused with 10% buffered formalin using transcardiac cauterization as previously described (11). The pigeon heads were placed in 10% EDTA in 10% buffered formalin (Sigma) that was changed twice a week. The progress of decalcification was monitored by weekly skull X-rays. After 30 days, the skulls were hemisected and blocked to include the labyrinths. Paraffin infiltration and embedding was performed on a Shandon Pathcentre instrument (Thermo Electron, Waltham, MA). The decalcified embedded and blocked tissue was serially sectioned at 6 µm and placed in triplicate on albuminized SuperFrost slides (Fisher Scientific). Subsequently, the sections on slides were deparaffinized and rehydrated by passage through xylene and graded ethanol solutions. The slides were then treated with 3% hydrogen peroxide with 0.03% sodium azide in PBS for 10 min. Next the slides were treated with protease XXIV (BioGenex, San Ramon, CA) for 8 min at room temperature. Following sequential 15-min incubations with 0.1% avidin and 0.01% biotin (Vector Laboratories, Burlingame, CA), to block endogenous avidin and biotin, the slides were incubated in 0.05% casein (Sigma)/0.05% Tween-20 (DAKO)/PBS for 30 min to block nonspecific protein binding. Primary rabbit polyclonal anti-Kir2.1 (no. APC-026; Alomone Labs, Jerusalem, Israel) was applied to sections at dilutions ranging from 1:90 to 1:120 for 60 min. Rabbit serum (InnoGenex, San Ramon, CA) was applied as a negative control. Biotinylated F(ab')2 fragments of swine anti- rabbit immunoglobulins (DAKO) supplemented with 10% pigeon serum served as the secondary antibody and was detected by streptavidin-HRP and colorized by DAB (DAKO). Slides were counterstained with Mayers modified hematoxylin (Poly Scientific, Bay Shore, NY) before mounting and viewed under an Olympus BX51 microscope, and images were recorded by an RT Slider Digital Camera (Diagnostic Instruments, Sterling Heights, MI).
Confocal Microscopy
CHO cells were grown on 25-mm coverslips in DMEM (Invitrogen/GIBCO) at 37°C, 75% humidity and 5% CO2. The cells were transiently transfected with a plasmid whose pKir2.1 and EGFP ORFs were under the control of the same promoter or with either of two fusion proteins: one with pKir2.1 in frame with EGFP but more proximal to the amino end of the protein; and the other with pKir2.1 more proximal to the carboxyl end. At 48 and 72 h posttransfection, the coverslips with plated cells were placed in a viewing chamber, and L-15 culture medium was substituted for the DMEM. The cells were imaged using a confocal microscope (Zeiss model LSM 510) using a Plan-Apochromat 63x/1.4 NA oil objective. The cells were optically scanned using a wavelength of 488 nm and optically sectioned at either 0.5 or 0.9 µm.
Bioinformatics
Sequence analysis was performed using two programs; DS Gene v1.15 (Accelrys, San Diego, CA) and Lasergene v5.07 (DNASTAR, Madison, WI). Probabilities that serine, threonine, and tyrosine were phosphorylation sites were obtained by submitting the ORF for pKir2.1 to the NetPhos 2.0 Server at the Center for Biological Sequence Analysis, Technical Univ. of Denmark (http://www.cbs.dtu.dk).
Data Analysis
Single-channel recordings were acquired and analyzed using either QuB (50, 51) or Clampex and Clampfit v9.0 (Axon Instruments) software. Whole cell macroscopic currents were acquired and analyzed using Clampex and Clampfit v9.0 (Axon Instruments). Data reduction was preformed using Origin v7.5 (OriginLab, Northampton, MA), PowerPoint 2002 (Microsoft, Redmond, WA), Photoshop v7.0 (Adobe, San Jose, CA), and Excel 2002 (Microsoft).
Throughout the text, unless otherwise specified, average results are expressed in the following format: mean ± standard error of the mean (SE), with number of samples (n). In the figures, error bars indicate ± 1 SE.
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RESULTS |
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Figure 2 shows the nucleotide sequence for pKir2.1 ORF cloned from total RNA from six semicircular canal ampullae (GenBank accession no. AF192507). The translated amino acids are shown above each nucleotide codon. The transmembrane domains M1 and M2 of the protein are indicated. Between domains M1 and M2 is the extracellular loop, the pore helix, and the pore domains. The M1-to-pore region and the pore-to-M2 region, comprising the extracellular loop domains, are demarked by parentheses and identified as MP and PM, respectively. Based on chimera studies (6) these regions have been identified as those involved in determining the open probability of single-channel responses of the Kir2.1 ion channel molecule. The highly conserved pore helix and pore regions are indicated by larger letters representing the amino acids. The pore helix region is demarked by square brackets.
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The signature amino acid motif (PKKR) for binding of phosphatidylinositol-4,5-bisphosphate (PIP2) is identified in Fig. 2 by a line above those amino acids that constitute the motif (amino acid 186 to 189). PIP2 binds directly to Kir channels (24). Bound PIP2 is necessary to activate Kir2.1 channels. In the Kir2.0 subfamily, the gating by PIP2 seems to depend only on the presence of PIP2 in the membrane (56, 68). The PKKR motif is highly conserved among members of the Kir2.0 subfamily.
To confirm that pKir2.1 is found in native hair cells, we conducted single cell RT-PCR using single hair cell cytoplasm from seven individual cells following patch clamp recording of current from the same cell. Figure 3A shows ionic currents recorded from a type II hair cell (cell C92). The test voltage protocol involved hyperpolarizing pulses from a negative holding potential of 63 mV. It can be seen that the peak amplitudes of the ionic currents increase with hyperpolarization and inactivate with time typical of IKir currents. Following acquisition of ionic currents, cytoplasm was extracted from the cells as shown in Fig. 3B, ac, and single cell RT-PCR was carried out. Figure 3C shows amplified product from 3/5 positive reactions (cells C66, C73, and C92) and 2/2 negative reactions (cells C91 and C93). It can be seen in Fig. 3, A and C, that cell C92 produced IKir currents and amplified product with a fragment size of 281 bp.
IKir and Ih are expressed with different frequencies in different regions of the ampullary and macular epithelium (39, 65). Moreover, the magnitudes of the currents are different in different regions of the epithelium. Figure 3D presents preliminary data suggesting that the expression and magnitude of IKir in a given cell in a given region may be transcriptionally regulated. Seventy cells were taken from different regions of the ampullary and utricular epithelium. Figure 3D results from an analysis of the cytoplasm from three of these cells. The cell that produced the band indicating 44 transcripts in its cytoplasm expressed an IKir current whose peak magnitude was 1.3 nA at a test voltage of 123 mV. On the other hand, cells showing four and seven pKir2.1 transcripts (lane 1 and lane 3), respectively, expressed no IKir current, only Ih (data not shown).
Table 2 summarizes a ClustalW alignment analysis of nucleotides in the ORF of pKir2.1 (AF192507) against comparable sequences for cIRK1 found in chick auditory papilla and Kir2.1 sequences from rhesus monkey lens, human midbrain, guinea pig heart, chick lens, and a mouse macrophage cell line. The cells of Table 2 contain the percent identity of the tissue referenced in the cell column and row. By viewing down the first column of Table 2, it can be seen that pKir2.1 ORF has high identity to the Kir2.1 subfamily of inwardly rectifying potassium channels across species and tissues.
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Figure 4A presents a phylogram relating the sequences shown in Table 2. If one views Fig. 4A from right to left, it can be seen that Kir2.1 channels found in avian epithelial cells (vestibular hair cell, auditory papilla, and lens epithelium) are clustered. This cluster then forms a distant node with the Kir2.1 inward rectifier sequence found in the mouse macrophage cell line (first cloned Kir2.1 sequence), which forms a subsequent node with Kir2.1 found in guinea pig heart cells. Finally, a node is formed between the Kir2.1 sequences in rodent cells and sequences from primate cells (rhesus monkey lens epithelium and human heart cells).
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Since the Kir2.1 ion channel protein determines a cells excitability by regulating the cells resting membrane potential, plays a role in regulation of K+ flux across the cell membrane, and is associated with the terminal repolarization phase of action potentials, its mRNA is found in many cell types and many tissues. Figure 5 presents a PAGE gel showing RT-PCR amplicons derived from total RNA isolated from cells of a variety of pigeon tissues. It is evident that pKir2.1 mRNA is found in all of the tissues tested.
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Figure 7 shows control sections (Fig. 7, A and C) and sections with reaction product in vestibular ganglion cells and in neck muscle (Fig. 7, B and D). This result complements the results shown in Fig. 5 indicating that Kir2.1 DNA (Fig. 5) and translated protein (Fig. 7) is in the vestibular nerve and neck skeletal muscle.
Figure 8 presents intracellular recordings from oocytes expressing pKir2.1. Figure 8A shows two-electrode voltage clamp traces to a voltage protocol shown above the current traces. The current traces in Fig. 8A-a show rapidly activating, rapidly inactivating currents in response to hyperpolarizing membrane potentials. The outward current in response to depolarizing voltages is highly attenuated. Figure 8A-b shows traces indicating the reduction in current during superfusion of the oocyte by a solution containing 100 µM BaCl2. Figure 8B is a current-voltage (I/V) plot indicating a partial block of steady-state current by 5 µM BaCl2 and a strong block by 100 µM BaCl2. The block relative to the control bath (ND96) almost completely recovered following washout of the BaCl2 by the control bath. Figure 8C indicates the sensitivity of the pKir2.1 current to K+ ions. When a ND96 solution containing 80 mM potassium gluconate was substituted for the control ND96 solution, the I/V curve shifted to the right and crossed the abscissa, 75 mV more depolarized. This shift in reversal potential is indicative of K+ sensitivity and is characteristic of the Kir2.1 subfamily of inward rectifiers. Figure 8D presents a comparison of IKir currents from a native hair cell from the utricle (Fig. 8D-a), an oocyte injected with pKir2.1 cRNA (Fig. 8D-b), and a CHO cell transfected with pKir2.1 DNA (Fig. 8D-c). When a monoexponential function was fitted to the inactivation of the currents, it became obvious that the oocyte expression system, probably because of its size (capacitance and resistance), was not a good model system to compare currents studied in this HES to those found in smaller native hair cells.
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DISCUSSION |
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Further verification of Kir2.0 subfamily membership of pKir2.1 using different techniques (genomics, electrophysiology, and immunocytochemistry) is the second novel feature of this paper. The ORF sequence for pKir2.1 (Fig. 2) shows high identity (>80%) with other Kir2.1 subfamily members in a variety of tissues and species, including primates (Table 2, Fig. 4A). Like other Kir2.1 subfamily members, there is considerable opportunity for posttranslational modulation of the ion channel by second messenger systems to ultimately control cell membrane potential (and therefore excitability), cellular K+ homeostasis, and gating of the channel itself. Fourteen of the 30 identified serine motifs have a 50% or greater chance of being active phosphorylation sites. The motifs for a PKA, a PKC, and a tyrosine kinase phosphorylation site are present. Four threonine and seven tyrosine motifs have a probability of 0.5 or greater of being a phosphorylation site. Kir2.1 channels are regulated by protein phosphorylation (9, 12, 22, 64, 66) and direct G protein activation (14, 26, 57). The inhibitory and facilitatory modulation probably fine tunes the cells excitability. There have been no studies of phosphorylation of Kir2.1 channels by protein kinase CK2. However, protein kinase CK2 has been shown to bind to and phosphorylate the carboxy termini of ENaC subunits (60) and ß-subunits of a voltage-dependent Ca2+ channel (30).
The ORF of pKir2.1 has the signature binding motif for PIP2. PIP2 is a second messenger that regulates the activity of a number of channels (13, 20). It has been shown to bind electrostatically (28) to positive base residues of Kir channels including Kir2.1 (24, 63, 68). PIP2 depletion causes IKir current rundown that can be reversed when PIP2 is restored. Recently, it has been shown (36) that a residue, R218, that is important in PIP2 binding when neutralized (R218/QW) is also associated with the channelopathy identified as Andersen syndrome (49). The presence of dysmorphic bodily features (seen in Andersen syndrome) suggests that, in addition to its role in cell excitability, K+ homeostasis and action potential repolarization, pKir2.1 might have a role in bodily feature development.
Table 2 and Fig. 4 suggest that there is a good deal of phylogenetic conservation of nucleotides carrying the message for IKir/Kir2.1. For example, there is 85% identity between the Kir2.1 ORFs (1,200 nucleotides) for pigeon hair cells and human heart cells. As expected, the phylogram (Fig. 4A) shows avian epithelial cells forming a node at one end of the phylogram and primate cells forming a node at the other end with rodents in between. Figure 4B extends the phylogenetic analysis of pKir2.1 and suggests that it is indeed a homomeric Kir2.1 subunit and not a pKir2.2, pKir2.3, or pKir2.4 subunit homomer or a mixed heteromer.
Kir2.1 is generally expressed in neurons, glial cells, and brain vasculature, but it is also expressed in smooth and skeletal muscle. Figure 5 shows a similar distribution of mRNAs encoding pKir2.1 in pigeon also with transcripts found in cell types and tissues as diverse as the sensory epithelia of the ear (hair cells from semicircular canals and utricle), epithelia from the eye (lens), neurons in the peripheral nervous system (vestibular ganglion cells), brain (brain stem, optic tectum and cerebellum), skeletal muscle (neck muscle), and cells from the heart and liver.
Figure 6 shows expression of pKir2.1 in the vestibular neuroepithelium, and Fig. 7 shows expression of pKir2.1 in vestibular ganglion cell bodies and neck muscle. In the vestibular epithelium, supporting cells and both types of hair cells (type I and type II) show immunoreactivity (see inset in Fig. 6D). It has previously been shown (39) from patch clamp studies that the primary current in pigeon vestibular supporting cells is IKir. IKir currents have been shown to be in type II hair cells in a number of species (4, 23, 39, 42, 58). The presence of IKir in type I vestibular hair cells is more controversial. Griguer et al. (18) recorded an inward rectifier current in type I hair cells in guinea pig. Subsequently, it was determined (54) that deactivation of a large outward rectifier current present in almost all pigeon type I hair cells (55) may have been mistakenly identified as the inward rectifier current. However, the present findings of Kir2.1 antibody immunoreactivity in type I hair cells suggest that pKir2.1 channels may be coexpressed in pigeon type I hair cells. Previous patch clamp studies (39, 65) have shown that IKir currents are regionally distributed in different zones on the pigeon vestibular epithelium. In the present study, there were no gradients of immunoreactivity in different areas of the epithelium (Fig. 6). It must be assumed that the antibody assay is too insensitive to detect the distributional variation of pKir2.1 channels in the epithelium as assayed by patch clamp recordings.
The traces in Fig. 1 point out several physiological roles for IKir channels in native pigeon hair cells. Those roles include setting the resting membrane potential and limiting membrane potential hyperpolarization, particularly when Ih is coexpressed. When pKir2.1 is blocked, the hair cells membrane potential depolarizes by 30 mV (moving it from near EK = 98 mV), and Ih is unmasked, causing a large hyperpolarization relative to the control response traces where both IKir and Ih are activated. It has been suggested (16, 17) that IKir has an additional physiological role in turtle auditory hair cells; that of contributing to hair cell tuning to different sound frequencies. The size of IKir varies inversely with the resonant frequency of the turtle auditory hair cell. Interestingly, in pigeon vestibular hair cells, resonant frequency is statistically significantly lower (
1/2) in regions of the epithelium (zone 3 of the crista and the ES zone of the utricle) where pKir2.1 is present and prominently expressed (65).
The reversal potential for Kir2.1 current shifts as EK shifts. As such, outward current flows only over a limited voltage range positive to EK regardless of the value of EK. Figure 8 illustrates this in oocytes transfected with pKir2.1. In Fig. 8C, the reversal potential shifts based on calculations using the Nernst equation. The membrane potential should have shifted 93 mV, but it actually shifted 87 mV. This difference was probably due to uncompensated leak current. No large shift occurred when 80 mM Na+ was washed onto the cell. It has been estimated that for Kir2.1 channels, the K+:Na+ selectivity is 1,500:1 (Ref. 53). Figure 8B shows another signature feature for Kir2.1 channels: Ba2+ sensitivity. In Fig. 8, 100 µM Ba2+ almost completely blocks the channel, yet 5 µM Ba2+ only blocks the channel by
15%. Previous work (61, 67) has shown that the EC50 for dose response curves for Kir2.1 channels is
2.5 µM. We carefully constructed a dose response curve (data not shown) for pKir2.1 expressed in CHO cells, and the EC50 was 3.7 µM.
From the cell-attached patch clamp data presented in Fig. 10, A and C, it can be seen that when using symmetrical 150 mM K+ solutions, the pKir2.1 channel activates at 30 mV and the inward current increases linearly over the range 30 to 150 mV. The mean slope conductance over this range is 29 pS. This value corresponds almost identically with other Kir2.1 channels [chick lens, 29 pS (52); and mouse macrophages, 30 pS (32)] as well as IKir channels [guinea pig ventricular cells, 27 pS (59); guinea pig alveolar cells, 31 pS (44); and rabbit osteoclasts, 31 pS (29)]. Figure 10B presents mean open and shut time for seven pigeon IKir experiments. Like mouse macrophage IKir expressed in oocytes (6), pKir2.1 single-channel recordings had single exponential open time distributions with mean open times ranging from 200300 ms, but unlike Kir2.1 expressed in oocytes (6) (which had 4 discrete closed states), pKir2.1 patches had a single exponential closed time distribution with mean closed times of 25 ms. The above results for pKir2.1 single-channel recording were based on two state recordings. However, it must be pointed out that like other Kir2.1 channels (52, 53) multiconductance states were frequently noticed.
The average resting membrane potential for type II hair cells in zone 3 of the pigeon crista (primarily pKir2.1 channels) is Vz = 68 mV (65). The average input resistance at that voltage is 1.5 G
(8); i.e., a total conductance (G) of 667 pS. These values were obtained in solutions where EK = 94 mV. In this paper, we report cloning pKir2.1 and measuring the single-channel conductance;
= 29 pS. Thus 23 pKir2.1 channels open all the time should be activated. But only 72% of the conductance at rest is for potassium since Vz = 68 mV not 94 mV. Thus 17 pKir2.1 channels are needed to account for the K+ conductance. However, the single-channel open probability at Vz = 68 mV is 0.650.8 (data not shown). Because of the open probability, 2126 pKir2.1 channels (84104 homotetrameric molecules of pKir2.1) are required to account for the conductance. The other currents IA, IK(Ca), ICa, and IK (coexpressed in pigeon type II vestibular hair cells) do not play a role in the above calculations since they do not activate until the membrane potential reaches at least 40 mV.
In conclusion, we have cloned a Kir2.0 subfamily channel in pigeon vestibular hair cells. We have concluded from phylogenetic analysis that it is the 2.1 subunit and not the other Kir2.0 subunits that is encoded in pigeon vestibular hair cells. We have shown that pKir2.1 has high ORF sequence identity with other members of the Kir2.1 subfamily in a variety of tissues from avians to humans. In pigeons, pKir2.1 transcripts are found in the vestibular epithelium, vestibular nerve, skeletal neck muscle, lens of the eye, heart, liver, and brain. Hair cells and supporting cells in the vestibular epithelium, as well as cells in the vestibular ganglion and neck muscle, immunoreact with Kir2.1 antibody, demonstrating pKir2.1 ion channel protein expression. We injected and transfected pKir2.1 cRNA and DNA into of oocytes and CHO cells, respectively. We carried out whole cell patch clamp electrophysiological studies and demonstrated that the K+ and Ba2+ sensitivities of the channels are the same as that reported for other Kir2.1 channels. Finally, we carried out cell-attached single-channel studies in CHO cells transfected with pKir2.1 and demonstrated that the single-channel features such as single-channel conductance and subconductance states were exactly like other Kir2.1 channels. The results obtained using all of the above techniques converge to the conclusion that the fast inward rectifier potassium channel in pigeon vestibular hair cells belongs to the Kir2.1 subfamily of inward rectifiers.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. J. Correia, Rm. 7.102, Blocker Medical Research Bldg., UTMB, Galveston, Texas 77555-1063 (E-mail: mjcorrei{at}utmb.edu).
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