Proton Block and Voltage Gating Are Potassium-dependent in the Cardiac Leak Channel Kcnk3
2000; Elsevier BV; Volume: 275; Issue: 22 Linguagem: Inglês
10.1074/jbc.m001948200
ISSN1083-351X
AutoresCoeli M. Lopes, Patrick G. Gallagher, Marianne E. Buck, Margaret H. Butler, Steven A. Goldstein,
Tópico(s)Neuroscience and Neural Engineering
ResumoPotassium leak conductances were recently revealed to exist as independent molecular entities. Here, the genomic structure, cardiac localization, and biophysical properties of a murine example are considered. Kcnk3 subunits have two pore-forming P domains and unique functional attributes. At steady state, Kcnk3 channels behave like open, potassium-selective, transmembrane holes that are inhibited by physiological levels of proton. With voltage steps, Kcnk3 channels open and close in two phases, one appears to be immediate and one is time-dependent (τ = ∼5 ms). Both proton block and gating are potassium-sensitive; this produces an anomalous increase in outward flux as external potassium levels rise because of decreased proton block. Single Kcnk3 channels open across the physiological voltage range; hence they are "leak" conductances; however, they open only briefly and rarely even after exposure to agents that activate other potassium channels. Potassium leak conductances were recently revealed to exist as independent molecular entities. Here, the genomic structure, cardiac localization, and biophysical properties of a murine example are considered. Kcnk3 subunits have two pore-forming P domains and unique functional attributes. At steady state, Kcnk3 channels behave like open, potassium-selective, transmembrane holes that are inhibited by physiological levels of proton. With voltage steps, Kcnk3 channels open and close in two phases, one appears to be immediate and one is time-dependent (τ = ∼5 ms). Both proton block and gating are potassium-sensitive; this produces an anomalous increase in outward flux as external potassium levels rise because of decreased proton block. Single Kcnk3 channels open across the physiological voltage range; hence they are "leak" conductances; however, they open only briefly and rarely even after exposure to agents that activate other potassium channels. two P domains and four predicted transmembrane segments base pair(s) kilobase(s) 4-morpholineethanesulfonic acid phorbol 12-myristate 13- acetate Leak currents are considered essential to normal electrical function in sympathetic ganglia (1.Jones S.W. Neuron. 1989; 3: 153-161Abstract Full Text PDF PubMed Scopus (66) Google Scholar, 2.Koyano K. Tanaka K. Kuba K. J. Physiol. 1992; 454: 231-246Crossref PubMed Scopus (22) Google Scholar), myelinated axons (3.Schmidt H. Stampfli R. Pfluegers Arch Gesamte Physiol. Menschen Tiere. 1966; 287: 311-325Crossref PubMed Scopus (51) Google Scholar, 4.Hille B. J. Gen. Physiol. 1973; 61: 669-686Crossref PubMed Scopus (317) Google Scholar, 5.Koh D.S. Jonas P. Brau M.E. Vogel W. J. Membr. Biol. 1992; 130: 149-162Crossref PubMed Scopus (54) Google Scholar, 6.Wu J.V. Rubinstein C.T. Shrager P. J. Neurosci. 1993; 13: 5153-5163Crossref PubMed Google Scholar), carotid body type 1 cells (7.Buckler K.J. J. Physiol. 1997; 498: 649-662Crossref PubMed Scopus (261) Google Scholar), and cardiac myocytes (8.Backx P.H. Marban E. Circ. Res. 1993; 72: 890-900Crossref PubMed Google Scholar, 9.Yue D.T. Marban E. Pfluegers Arch. Eur. J. Physiol. 1988; 413: 127-133Crossref PubMed Scopus (85) Google Scholar, 10.Van Wagoner D.R. Pond A.L. McCarthy P.M. Trimmer J.S. Nerbonne J.M. Circ. Res. 1997; 80: 772-781Crossref PubMed Scopus (498) Google Scholar, 11.Boyle W.A. Nerbonne J.M. J. Gen. Physiol. 1992; 100: 1041-1067Crossref PubMed Scopus (74) Google Scholar, 12.Wang Z. Fermini B. Nattel S. Circ. Res. 1993; 73: 1061-1076Crossref PubMed Scopus (513) Google Scholar). Nonetheless, their existence as independent transport entities, rather than residual flux through other pathways, was controversial until the cloning of KCNKØ from Drosophila melanogaster (13.Goldstein S.A.N. Price L.A. Rosenthal D.N. Pausch M.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13256-13261Crossref PubMed Scopus (166) Google Scholar). KCNKØ (previously ORK1), encodes a potassium channel subunit with two P domains and four predicted transmembrane segments (2P/4TM)1 (13.Goldstein S.A.N. Price L.A. Rosenthal D.N. Pausch M.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13256-13261Crossref PubMed Scopus (166) Google Scholar). KCNKØ channels are open across the physiological voltage range, show no delay in current development with voltage steps, and "openly rectify," that is, they operate like potassium-selective holes in an electric field (13.Goldstein S.A.N. Price L.A. Rosenthal D.N. Pausch M.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13256-13261Crossref PubMed Scopus (166) Google Scholar). 2N. Ilan and S. A. N. Goldstein, submitted for publication. 2N. Ilan and S. A. N. Goldstein, submitted for publication. KCNKØ channels are tightly regulated; activation yields an open probability (P o) close to 1, and inhibition produces channels that are almost always closed (63.Ilan N. Zilberberg N. Gonzalez-Colaso R. Goldstein S.A.N. Biophys. J. 1999; 76 (abstr.): 411Google Scholar). 3N. Zilberberg, N. Ilan, R. Gonzalez-Colaso, and S. A. N. Goldstein, submitted for publication. 3N. Zilberberg, N. Ilan, R. Gonzalez-Colaso, and S. A. N. Goldstein, submitted for publication. Mammalian genes homologous to KCNKØ, now enumerated KCNK1–9, are emerging rapidly. Like KCNKØ, those that show function are potassium-selective leak conductances (16.Goldstein S.A.N. Wang K.W. Ilan N. Pausch M. J. Mol. Med. 1998; 76: 13-20Crossref PubMed Scopus (74) Google Scholar, 17.Fink M. Lesage F. Duprat F. Heurteaux C. Reyes R. Fosset M. Lazdunski M. EMBO J. 1998; 17: 3297-3308Crossref PubMed Scopus (394) Google Scholar, 18.Reyes R. Duprat F. Lesage F. Fink M. Salinas M. Farman N. Lazdunski M. J. Biol. Chem. 1998; 273: 30863-30869Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 19.Pountney D.J. Gulkarov I. de Miera E.V. Holmes D. Saganich M. Rudy B. Artman M. Coetzee W.A. FEBS Lett. 1999; 450: 191-196Crossref PubMed Scopus (54) Google Scholar, 20.Salinas M. Reyes R. Lesage F. Fosset M. Heurteaux C. Romey G. Lazdunski M. J. Biol. Chem. 1999; 274: 11751-11760Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 21.Colquhoun D. Sigworth F.J. Sakmann B. Neher E. Single-channel Recording. 2nd Ed. Plenum Press, New York1995: 483-588Crossref Google Scholar, 22.Chavez R.A. Gray A.T. Zhao B.B. Kindler C.H. Mazurek M.J. Mehta Y. Forsayeth J.R. Yost C.S. J. Biol. Chem. 1999; 274: 7887-7892Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 23.Kim Y. Bang H. Kim D. Biophys. J. 2000; 78 (abstr.): 207Google Scholar, 24.Lopes C.M.B. Gallagher P.G. Wong C. Buck M. Goldstein S.A.N. J. Biophys. 1998; 74 (abstr.): 44Google Scholar). Based on homology to KCNKØ we isolated Kcnk3 from a murine cardiac cDNA library (16.Goldstein S.A.N. Wang K.W. Ilan N. Pausch M. J. Mol. Med. 1998; 76: 13-20Crossref PubMed Scopus (74) Google Scholar, 24.Lopes C.M.B. Gallagher P.G. Wong C. Buck M. Goldstein S.A.N. J. Biophys. 1998; 74 (abstr.): 44Google Scholar), localized it to chromosome 5B in mouse and 2p23.3-p24.1 in human (25.Manjunath N.A. Bray-Ward P. Goldstein S.A.N. Gallagher P.G. Cytogen. Cell Gen. 1999; 86: 242-243Crossref PubMed Google Scholar), and called the predicted protein product OAT1. Two other groups cloned Kcnk3 concurrently and called the encoded subunit TASK1 (26.Duprat F. Lesage F. Fink M. Reyes R. Heurteaux C. Lazdunski M. EMBO J. 1997; 16: 5464-5471Crossref PubMed Scopus (542) Google Scholar) and TBAK1 (27.Kim D. Fujita A. Horio Y. Kurachi Y. Circ. Res. 1998; 82: 513-518Crossref PubMed Scopus (113) Google Scholar). For clarity, we will now employ the Human Genome Organization nomenclature:KCNK3 gene and KCNK3 channel for human isolates andKcnk3 and Kcnk3 for mice. Significant discrepancies exist between the findings of the three groups. Although all agree thatKcnk3 predicts 2P/4TM subunits that form pH-sensitive, openly rectifying potassium channels, there is no consensus as to tissue distribution (atria or ventricle), function (instantaneous or time-dependent, low or high open probability), or the predicted protein sequence. In this report, five points are highlighted. First, the genomic sequence for murine Kcnk3 is determined to confirm the accuracy of the cDNA under study; this reveals an intron in the midst of the coding sequence for the signature motif (G YG) of the first P loop (an arrangement seen to be conserved in the family from nematodes to humans). Second, Kcnk3 messenger RNA is localized to murine cardiac ventricle and, at lower levels, in the atria; Kcnk3 protein is then confirmed to have the same cardiac distribution. Third, half-maximal blockade of Kcnk3 channels by external protons is confirmed to be near physiological pH and shown to be sensitive to external potassium. Fourth, Kcnk3 currents are seen to develop with voltage changes in two phases; one appears to be immediate and one is time-dependent; the fraction of current in each phase is responsive to external potassium. Fifth, single Kcnk3 channels are shown to open only briefly (to one of two conductance levels) and rarely; although open probability increases with depolarization, it is not significantly augmented by a wide array of stimuli including activation or inhibition of protein kinase A or C, application of volatile anesthetics or metabolic poisons, changes in osmotic strength, or exposure to low oxygen tension. Based on its location and similar functional attributes, we hypothesize Kcnk3 to be the correlate of a native cardiac current that remains active throughout the action potential plateau but whose molecular basis has been unclear, I Kp orI Ksus (8.Backx P.H. Marban E. Circ. Res. 1993; 72: 890-900Crossref PubMed Google Scholar, 9.Yue D.T. Marban E. Pfluegers Arch. Eur. J. Physiol. 1988; 413: 127-133Crossref PubMed Scopus (85) Google Scholar, 10.Van Wagoner D.R. Pond A.L. McCarthy P.M. Trimmer J.S. Nerbonne J.M. Circ. Res. 1997; 80: 772-781Crossref PubMed Scopus (498) Google Scholar, 11.Boyle W.A. Nerbonne J.M. J. Gen. Physiol. 1992; 100: 1041-1067Crossref PubMed Scopus (74) Google Scholar, 12.Wang Z. Fermini B. Nattel S. Circ. Res. 1993; 73: 1061-1076Crossref PubMed Scopus (513) Google Scholar, 28.Apkon M. Nerbonne J.M. Biophys. J. 1988; 53 (abstr.): 458Google Scholar). The findings support the idea that Kcnk3 channels link cardiac excitability to changes in acid-base status. A homology search of NCBI data base using the BLAST program suite (29.Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69652) Google Scholar) and the coding sequence of the ORK1 channel (KCNKØ) as query (accession number U55321) identified expressed sequence tag W09160. Northern blot analysis using the 801-bp cDNA fragment in W09160 detected an abundant single message at ∼3.8 kb in murine heart (24.Lopes C.M.B. Gallagher P.G. Wong C. Buck M. Goldstein S.A.N. J. Biophys. 1998; 74 (abstr.): 44Google Scholar). The 801-bp cDNA fragment was used to screen a random primed and oligo(dT)-primed murine heart cDNA library in λγt11 (CLONTECH, Palo Alto, CA). Of 28 clones that hybridized to the probe, eight were purified and subcloned, and their ends subjected to automated DNA sequencing; three clones were sequenced in their entirety. This yielded a 5′-untranslated sequence, an open reading frame, and a 3′-untranslated sequence. An additional 160 bp of the upstream 5′-untranslated sequence was obtained by 5′ rapid amplification of cDNA ends using 1 μg of total RNA prepared from murine cardiac muscle, as described (30.Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63084) Google Scholar) and primer A (5′-CACCAGCAGGTAGGTGAAG-3′). Single-stranded ligation and amplification were carried out with primers D (5′-GCCAAGCTTGCGGTGGCCCTCAGGTCCAGCTC-3′) with B (5′-GCCGCCGCTGCTGCCCCGGA-3′) and D with C (5′-CTCCACGCCGGGCACCAGCTCCGCAC-3′), respectively. Analyses of nucleotide and predicted amino acid sequences were performed utilizing GCG software from the University of Wisconsin (Madison, WI). The cDNA sequence is listed (accession number AB008537). The coding sequence was placed between the 5′- and 3′-untranslated regions the ofXenopus β-globin gene in pBF2 (a gift of Bernd Fakler, Tuebingen, DE) and cRNA produced using T3 RNA polymerase and a kit (Ambion, Austin, TX). Transcripts were quantified by spectrophotometer and compared with control samples separated by agarose gel electrophoresis. Our cDNA sequence (accession number AF065162) was verified by comparison with the genomic sequence (accession numbersAF241798 and AF242508) and varies from the partial sequence reported for murine TASK1 at amino acid residues 4 (Gln replaces Glu), 123 (Val replaces Ile), and 286 (where an additional Gly is added) (26.Duprat F. Lesage F. Fink M. Reyes R. Heurteaux C. Lazdunski M. EMBO J. 1997; 16: 5464-5471Crossref PubMed Scopus (542) Google Scholar) and murine TBAK1, which includes a 9-residue amino-terminal extension and a single residue difference at position 101 (Pro replaces Ala) (27.Kim D. Fujita A. Horio Y. Kurachi Y. Circ. Res. 1998; 82: 513-518Crossref PubMed Scopus (113) Google Scholar). These differences do not coincide with known consensus sites in theKcnk3 genomic clone (see below) for splice junctions or editing and are judged to be errors in the sequences reported by others. A murine genomic DNA library in bacteriophage P1 was screened with two oligonucleotide primers corresponding to the 3′ end of the coding region of Kcnk3 cDNA as described (31.Pierce J.C. Sternberg N. Sauer B. Mamm. Genome. 1992; 3: 550-558Crossref PubMed Scopus (76) Google Scholar). These primers, 5′-GCAGACGCAGCCGCAGTATG-3′ and 5′-GCCTGGCCGTTGTGCGTGAGCAGGG-3′, amplify a 168-bp fragment from murine genomic DNA. Polymerase chain reaction-positive clones were purified and subcloned into pGEM-7Z plasmid vectors (Promega Corp., Madison, WI). Subcloned fragments were analyzed by restriction endonuclease digestion, Southern blotting, and nucleotide sequencing. 32P[ATP]-labeled probes used for Northern blots were the 801-bp fragment (W09160) and a β-actin cDNA (Amersham Pharmacia Biotech) (32.Ng S.Y. Gunning P. Eddy R. Ponte P. Leavitt J. Shows T. Kedes L. Mol. Cell. Biol. 1985; 5: 2720-2732Crossref PubMed Google Scholar). In situ hybridization was performed with adult C57BL6 mice (Jackson Labs, Bar Harbor, ME) using sense and antisense probes from the 801-bp Kcnk3 fragment, as described (33.Reppert S.M. Weaver D.R. Stehle J.H. Rivkees S.A. Mol. Endocrinol. 1991; 5: 1037-1048Crossref PubMed Scopus (294) Google Scholar). [α-35S]UTP (Amersham Pharmacia Biotech) incorporation into the 801-bp Kcnk3 fragment was 70–85%. Heart sections (8 μm) were hybridized overnight, treated with ribonuclease-A, and dehydrated by soaking in 100% EtOH in 0.6m ammonium acetate. Emulsion radiographs were generated by dipping slides in photographic emulsion with development and fixation 2 days later. Slides were placed on Kodak SB5 film to generate images. Rabbit antibodies recognizing residues 252–269 of the human KCNK3 subunit, EDEKRDAEHRALLTRNGQ, were purchased from Alamone (APC024, Jerusalem, Israel). Frozen mouse heart ventricle and atria were purchased (Pel-Freez Biologicals, Rogers, AZ), and crude membrane fractions of each tissue were prepared by a modified method (34.Kandror K.V. Coderre L. Pushkin A.V. Pilch P. Biochem. J. 1995; 307: 383-390Crossref PubMed Scopus (95) Google Scholar). Proteins were extracted with 1% Triton X-100 and analyzed by SDS-polyacrylamide gel electrophoresis and Western blot with the rabbit antibody, followed by goat anti-rabbit horseradish peroxidase-conjugated antibody and visualization by enhanced chemiluminescent substrate. Oocytes were isolated from Xenopus laevis frogs (Nasco, Atkinson, WI), subjected to collagenase treatment to ease removal of the follicle, and injected with 46 nl of sterile water containing 2–4 ng of Kcnk3 cRNA. Macroscopic currents were measured 1–4 days after cRNA injection by two-electrode voltage clamp using a Geneclamp 500 amplifier (Axon Instruments, Foster City, CA). Data were sampled at 4–20 kHz and filtered at 1–5 kHz. Data acquisition and analysis were performed using Pulse (Instrutech, Great Neck, NY) and Sigmaplot (Jandel Scientific, San Rafael, CA) software. Electrodes were made from 1.5-mm borosilicate glass tubes (Garner Glass Co., Claremont, CA), contained 3 m KCl, and had resistances between 0.3 and 1 MΩ. Oocytes were studied while perfused at 0.5–1 ml/min with 5 mm KCl bath solution 93 mm NaCl, 5 mm KCl, 1 mm MgCl2, 0.3 mm CaCl2, 5 mm HEPES, pH 7.4, with NaOH. In indicated cases, KCl was substituted for NaCl. For solutions at pH 6.0, MES replaced HEPES. Studies were performed at room temperature. Voltage clamp recordings were made in both on-cell and outside-out configuration using an Axopatch 200A amplifier (Axon Instruments). The vitelline layer was removed prior to recording with a pair of fine forceps after a 1–2-min incubation in hypertonic solution 200 mm potassium aspartate, 20 mm KCl, 1 mm MgCl2, 10 mm EGTA, 10 mm HEPES, pH 7.4, with NaOH. Pipettes were fabricated from 7052 glass (Garner Glass Co., Claremont, CA) coated with Q-Dope (GC Electronics, Rockford, IL) and fire-polished. The electrode solution for outside-out patches was 100 mm KCl, 5 mmEGTA, 1 mm MgCl2, 5 mm HEPES, pH 7.4, with KOH. Bath solution contained 100 mm KCl, 0.3 mm CaCl2, 1 mm MgCl2, 5 mm HEPES, pH 7.4. External potassium concentration was varied by substitution of NaCl for KCl. Pipette resistance ranged from 3–5 MΩ for single channel recordings and 0.3–0.6 MΩ for macropatches; seal resistance was 4–15 GΩ. Data were sampled at 10–40 kHz, filtered at 0.5–5 kHz with ACQUIRE software (Instrutech Corp.), and analyzed off-line by TAC (Instrutech Corp.) and IGOR (Wavemetrics, Lake Oswego, OR) software. Equilibrium reversal potentials were determined in the indicated solutions by linear regression. Current-voltage relations were studied in various potassium solutions and fit to the Goldman (35.Goldman D.E. J. Gen. Physiol. 1943; 27: 37-60Crossref PubMed Scopus (1667) Google Scholar) and Hodgkin and Katz (36.Hodgkin A.L. Katz B. J. Physiol. 1949; 108: 37-77Crossref PubMed Scopus (1806) Google Scholar) current relationships. IS=PKzS2VF2RT[K] i−[K] o exp(−zS VF/RT)1−exp(−zS VF/RT)Equation 1 where P K is the permeability of potassium, z, V, F, R, andT have their usual meanings, and an internal K+concentration of 90 mm is assumed, as reported previously (37.Wang K.-W. Tai K.-K. Goldstein S.A.N. Neuron. 1996; 16: 571-577Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Permeability ratios were calculated according to the following equation. PKPX=exp −Vrev FRTEquation 2 where P K and P Xare the permeability of potassium and the test cation, respectively; in whole cell mode it is assumed that potassium is the only permeant ion inside the cell. Dose response curves were fit to the following function. 11+[B]K1/2nEquation 3 where [B] is the concentration of the blocker,K 1/2 is the concentration of the blocker required to achieve 50% inhibition, and n is the Hill coefficient. The voltage dependence of block was modeled by a simplification of the approach of Woodhull (38.Woodhull A.M. J. Gen. Physiol. 1973; 61: 687-708Crossref PubMed Scopus (1232) Google Scholar) according to the following equation. IImax=[B]K1/2exp−zδFVRT+1−1Equation 4 where z and δ represent charge on the blocker and the apparent electrical distance traversed by the blocking particle to reach its receptor site. Three independent cDNA clones forKcnk3 isolated from a murine cardiac library were found to be identical (accession number AF065162) (24.Lopes C.M.B. Gallagher P.G. Wong C. Buck M. Goldstein S.A.N. J. Biophys. 1998; 74 (abstr.): 44Google Scholar). Relative to the predicted initiator methionine, the cDNA contains an A in position −3 and a termination codon 209 bp upstream with no additional ATG triplets in the intervening sequence. The open reading frame is 1227 bp, and secondary structure analyses predict a protein of 409 amino acids with two classical P domains (2P) bounded by hydrophobic segments that suggest the presence of four transmembrane segments (4TM) (Fig.1 A). A 2P/4TM topology is consistent with the absence of a recognizable leader sequence, the external disposition of one consensus site for N-linked glycosylation, and an internal location for two sites for protein kinase C, two for protein kinase A, three for calcium-calmodulin kinase II, one for tyrosine kinase, and a carboxyl-terminal PDZ consensus motif. Alignments of the P domains for subunits predicted to have two P domains reveals that Kcnk3 is most similar to the open and outward rectifiers (KCNKØ, KCNK2, Kcnk4, and KCNK5), more distantly related to clones with as yet undefined function (KCNK1, Kcnk6, Kcnk7, and KCNK8) and most distinct from the outward rectifier of yeast cells, TOK1 (Fig.1 B). Homology among the genes is insignificant except for the P domain segments where pairs can achieve ∼30% identity. We verified the predicted Kcnk3 cDNA sequence by comparison to the genomic DNA sequence (accession numbers AF241798 andAF242508). Three Kcnk3 genomic clones were identified by polymerase chain reaction screening, and one was studied in detail by restriction enzyme analysis, Southern blotting, and limited nucleotide sequencing. The region of this clone containing the Kcnk3gene, including 5′and 3′-untranslated sequences and the coding region, were sequenced on both strands. Comparison of cDNA and genomic sequences showed that Kcnk3 is a two exon gene spread over ∼21 kb (Fig. 1 C). Evaluation of the exon/intron boundaries revealed the AG:GT rule was not violated and that no AG nucleotide pairs were present in the 15 bp upstream of the 3′ (acceptor) splice junction. The single exon/intron boundary is located at a functionally critical position in the channel, in the midst of the selectivity filter "signature sequence" (G YG) of the first P domain. Northern blot analysis using an 801-bp Kcnk3cDNA fragment as probe detected a strong signal in heart with less abundant message in lung and brain (Fig.2 A). Only a single band at ∼3.8 kb was detected. Faint signals were visualized after exposure for extended periods in skeletal muscle and kidney (not shown). When the distribution of Kcnk3 message in mouse heart was examined by in situ hybridization, a strong, specific signal was apparent throughout both ventricles with the antisense probe (Fig.2 C). A weak antisense signal was also visualized in the atria, indicating a lower level of transcript in those cells. Because this localization was at odds with prior reports (26.Duprat F. Lesage F. Fink M. Reyes R. Heurteaux C. Lazdunski M. EMBO J. 1997; 16: 5464-5471Crossref PubMed Scopus (542) Google Scholar), we evaluated the cardiac expression pattern of Kcnk3 protein. Anti-peptide antibodies were used to visualize Kcnk3 protein in homogenates of murine atrial and ventricular tissue (Fig. 2 D). A strong signal near the predicted mass for Kcnk3 was apparent in ventricular samples (Fig. 2 D, lane 1); a weaker signal was found in atrial preparations despite the presence of similar amount of total protein in the lane (Fig. 2 D, lane 2). The signal was demonstrated to be specific for Kcnk3 protein because it was competitively depleted by co-incubation with the peptide fragment recognized by the antiserum (Fig. 2 D, lanes 3 and4). When Kcnk3 cRNA was injected into X. laevis oocytes, a new current was observed by two-electrode voltage clamp (Fig. 3 A) that was not present in control cells. In response to changes in voltage, the current rose to a new steady state level. Once activated, inactivation was not observed (10 s pulses; not shown). At physiological levels of bath potassium (5 mm) and pH (7.4), the channel passed large outward currents with depolarizing voltage steps but only small inward currents at hyperpolarized potentials (Fig.3 A, left panel). Increasing external potassium concentration produced a shift in reversal potential and large inward currents (Fig. 3, A and B). The change in reversal potential indicated that the channel was selective for potassium over sodium and chloride. Thus, increasing external potassium levels from 5 to 100 mm (by isotonic substitution of NaCl with KCl) produced a shift in reversal potential of 56 ± 3 mV/10-fold change in potassium (Fig. 3 C) in good agreement with the Nernst relation, which predicts an ∼58 mV change for a perfectly selective channel under these conditions. Changes in current-voltage relationships with altered external potassium indicated that Kcnk3 channels were openly rectifying. Thus, inward currents were smaller than outward currents when external potassium was low and increased to equal magnitude when potassium levels were approximately the same across the membrane (Fig. 3,A and B). This behavior was reasonably well approximated by the Goldman-Hodgkin-Katz relation (Equation 1) for current through across an ion-selective partition at differing transmembrane gradients of permeant ion (Fig. 3 B). It was notable that Equation 1 failed to approximate the experimental data at 5 mm bath potassium because outward currents were smaller than predicted. This was subsequently explained by potassium-dependent proton inhibition of Kcnk3 channel currents (see below). Kcnk3 channels exhibit an Eisenman type III permeability series (Fig.3 D). To assess relative permeability compared with potassium (the predominant internal permeant ion), a test cation replaced the sodium and potassium in the bath solution to achieve a pseudo bi-ionic condition in whole cell mode and Equation 2 was used. Permeability was highest for rubidium (1.1 ± 0.1, n = 12) and potassium (= 1), intermediate for cesium and ammonium (0.30 ± 0.02 and 0.23 ± 0.03, n = 8, respectively), and lowest for sodium and lithium (less than 0.031 ± 0.003 and 0.031 ± 0.002, n = 8, respectively). Although rubidium had a greater relative permeability than potassium its relative conductance was over 2-fold lower (Fig. 3 D). With 5 mm potassium in the bath, Kcnk3 currents were maximal at pH 8.0, significantly blocked at pH 7.4, and completely inhibited at pH 6.0 (Fig.4 A). Proton block was well fit to Equation 3 with a half-maximal blocking concentration (pK a) of 7.24 ± 0.03 and a Hill coefficient of 1.02 ± 0.06, suggesting that one proton was required to block (Fig. 4 B). As external potassium levels rose, block by protons was diminished (Fig. 4 C); at pH 7.0, the fraction of unblocked current at 30 mV in a potassium-free bath solution was 0.32 ± 0.02 and increased to 0.54 ± 0.02, 0.73 ± 0.02 and 0.94 ± 0.02 with 5, 20, and 100 mm bath potassium, respectively. Increasing proton levels inhibited Kcnk3 channels despite elevated potassium levels (not shown). The effect of potassium on proton inhibition explained the anomalous increase in outward current seen with elevation of external potassium (Fig.3 A); although increasing bath potassium decreased the outward driving force for potassium flux, it also diminished proton inhibition (Fig. 4 C) leading to an overall increase in outward current. The rise and fall of Kcnk3 currents showed a phase that appeared immediate and another that was delayed. In whole cell mode, ∼40% of activation was judged to be time-dependent with steps from −80 to 60 mV at physiological levels of pH (7.4) and potassium (5 mm) (Fig.5 A). Both raising external potassium from 5 to 100 mm (Fig. 5 B, left panel) and decreasing protons from pH 7.0 to 8.0 (Fig.5 A, right panel) decreased the fraction of current that was time-dependent (I TD/I). Similarly, 55% of deactivating current was judged to be time-dependent with a step from 60 to −120 mV at pH 7.4 and 5 mm potassium (Fig.5 C), and raising external potassium (Fig. 5 D,left panel) and lowering proton level (Fig. 5 D,right panel) decreased the fraction of current that was time-dependent. I TD/Ireflects the fraction of channels closed at rest; thus, higher potassium and lower proton in the bath increased the fraction of channels that were open before the test pulse. At physiological resting potentials and ionic conditions, roughly half the Kcnk3 channels that passed current upon depolarization were already open. The effects of external potassium were consistent with the theory that increased occupancy of the external pore by potassium (either by raising bath levels or decreasing proton block) favored the open channel state. Kcnk3 currents in on-cell patches showed changes with voltage consistent with altered open probability (Fig.6). Thus, the fraction of time-dependent current decreased with more positive holding potential (from −150 to −60 mV; Fig. 6 A, middle panel) or test pulse (from 0 to 60 mV; Fig. 6 A,right panel), indicating opening of channels by depolarization. Deactivation showed a similar dependence on voltage; activation at positive potentials (from 0 to 60 mV; Fig. 6 B,middle panel) opened more channels, increasing the fraction of current that decayed upon subsequent hyperpolarization, and more positive deactivation potential (from -150 to -60 mV; Fig.6 B, right panel) decreasedI TD/I, presumably by increasing the number of channels that stayed open. Activation and deactivation were well approximated by single exponential relationships, and both rates showed a weak dependence on voltage. The rate of activation was greater at more positive test potentials (Fig. 6 B), although deactivation was faster at more negative voltages (Fig. 6 D). With 5 mm external potassium at pH 7.4, the time constant (τ) for the rise in the current (with a step from −80 to 45 mV) was 4.4 ± 0.5 ms and showed an e-fold change per ∼250 mV over this voltage range (Fig. 6 B). This rate of was largely insensitive to both external potassium and pH. Thus, τ with 20 mm potassium at pH 7.4 was 4.1 ± 0.2 ms (n = 3), whereas it was 4.4 ± 0.4 and 4.3 ± 0.6 ms at 5 mm potassium at pH 8.0 (n = 4) and pH 7.0 (n = 4), resp
Referência(s)