KT3.2 and KT3.3, Two Novel Human Two-Pore K+ Channels Closely Related to TASK-1

Eleazar Vega-Saenz de Miera,1 David H. P. Lau,1 Maria Zhadina,1 David Pountney,2 William A. Coetzee,1,2 and Bernardo Rudy1,3

 1Department of Physiology and Neuroscience,  2Department of Pediatric Cardiology, and  3Department of Biochemistry, New York University School of Medicine, New York, New York 10016


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Vega-Saenz de Miera, Eleazar, David H. P. Lau, Maria Zhadina, David Pountney, William A. Coetzee, and Bernardo Rudy. KT3.2 and KT3.3, Two Novel Human Two-Pore K+ Channels Closely Related to TASK-1. J. Neurophysiol. 86: 130-142, 2001. We report the cloning of human KT3.2 and KT3.3 new members of the two-pore K+ channel (KT) family. Based on amino acid sequence and phylogenetic analysis, KT3.2, KT3.3, and TASK-1 constitute a subfamily within the KT channel mammalian family. When Xenopus oocytes were injected with KT3.2 cRNA, the resting membrane potential was brought close to the potassium equilibrium potential. At low extracellular K+ concentrations, two-electrode voltage-clamp recordings revealed the expression of predominantly outward currents. With high extracellular K+ (98 mM), the current-voltage relationship exhibited weak outward rectification. Measurement of reversal potentials at different [K+]o revealed a slope of 48 mV per 10-fold change in K+ concentration as expected for a K+-selective channel. Unlike TASK-1, which is highly sensitive to changes of pH in the physiological range, KT3.2 currents were relatively insensitive to changes in intracellular or extracellular pH within this range due to a shift in the pH dependency of KT3.2 of 1 pH unit in the acidic direction. On the other hand, the phorbol ester phorbol 12-myristate 13-acetate (PMA), which does not affect TASK-1, produces strong inhibition of KT3.2 currents. Human KT3.2 mRNA expression was most prevalent in the cerebellum. In rat, KT3.2 is exclusively expressed in the brain, but it has a wide distribution within this organ. High levels of expression were found in the cerebellum, medulla, and thalamic nuclei. The hippocampus has a nonhomogeneous distribution, expressing at highest levels in the lateral posterior and inferior portions. Medium expression levels were found in neocortex. The KT3.2 gene is located at chromosome 8q24 1-3, and the KT3.3 gene maps to chromosome 20q13.1.


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ABSTRACT
INTRODUCTION
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DISCUSSION
REFERENCES

Potassium channels are ubiquitously expressed and play critical roles in both excitable and nonexcitable cells. They help to determine the resting potential as well as the excitability and firing properties of excitable cells (Hille 1992; Llinas 1988; Rudy 1988). A large number of mammalian potassium-channel subunits have been described (Chandy and Gutman 1993; Coetzee et al. 1999; Isomoto et al. 1997; Jan and Jan 1997). The latest addition to this expanding set of mammalian K+-channel subunits is a group of subunits containing two separate pore regions within a single subunit. The first prototypes of this group YOK1/TOK1 was discovered in yeast (Ketchum et al. 1995; Lesage et al. 1996a) and consist of subunits containing eight transmembrane domains (TMDs) and two pore regions. K+-channel subunits having two pore regions within a single polypeptide (KT) have also been described in mammals. However, all of these subunits described to date contain only four TMDs (termed M1-M4) with the two pore regions (P1 and P2), respectively, located between TMDs 1 and 2 and between TMDs 3 and 4. K+-channel subunits having this last structural organization have been identified in invertebrates (Goldstein et al. 1996; Wei et al. 1996), mammals (Chavez et al. 1999; Duprat et al. 1997; Fink et al. 1996, 1998; Lesage et al. 1996b; Pountney et al. 1999; Reyes et al. 1998; Salinas et al. 1999), and plants (Czempinski et al. 1997), suggesting a common ancestral origin before the evolutionary diversification of plants and animals. Seven genes of the KT family have been identified in mammals. In the order of their discovery, they are: TWIK1 (Lesage et al. 1996b), TREK (Fink et al. 1996), TASK-1 (Duprat et al. 1997; Leonoudakis et al. 1998), TRAAK/KT4.1 (Fink et al. 1998; A. Ozaita and E. Vega-Saenz de Miera, unpublished data), TASK-2 (Reyes et al. 1998), TWIK-2/TOSS (Chavez et al. 1999; Pountney et al. 1999), and KCNK6/KCNK7 (Salinas et al. 1999).

Here, we describe the cloning of two new members of this emerging group of mammalian K+-channel subunits, human KT3.2 and KT3.3, having a sequence similarity closest to TASK-1. KT3.2 subunits express channels that differ from TASK-1 channels in pH sensitivity, modulation by PMA, and tissue distribution.

A portion of these results was presented at the 44th annual meeting of the Biophysical Society.


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Bioinformational analysis

Initial identification of the sequences was obtained by using the Blast algorithm (http://www.ncbi.nlm.nih.gov/blast/blast.cgi). Exon identification was performed by using Blast of two sequences (http://www.ncbi.nlm.nih.gov/gorf/bl2.html), followed by sequence translation (http://www.expasy.ch/tools/dna.html) and identification of sequences by comparison to the amino acid sequence of human TASK-1 and finding the consensus sequence for intron/exon boundaries (Stephens and Schneider 1992). Hydropathy analysis was performed using the Kyte and Doolittle algorithm (Kyte and Doolittle 1982) implemented in ProtScale web page (http://www.expasy.ch/cgi-bin/protscale.pl). The prediction of phosphorylation sites and posttranslational modifications analysis was performed using ProfileScan (http://www.isrec.isb-sib.ch/software/PFSCAN_form.html).

Cloning of human KT3.2

Forward and reverse primers to amplify the full coding sequence of KT3.2 by polymerase chain reaction, using human brain cDNA (Clontech) as template, were designed by comparison of a human genomic sequence (Accession No. AC007869) and the sequence of human TASK-1 (Duprat et al. 1997). The sequence for the forward primer was (TTG CTG GCG GCC ATG AAG AG) and for the reverse primer was (CTA AAC GGA CTT CCG GCG TTT C). The PCR conditions were: 35 cycles, 54°C annealing, 72°C extension, and 94°C denaturation, each step for 1 min. The band was subcloned into pCR2.1 using TA cloning (Invitrogen), and both strands were sequenced.

To obtain additional 5' UTR sequences, rapid amplification of cDNA ends (RACE) experiments were performed using nested primers. The first round of amplification was performed using a specific oligonucleotide (AAC CTA TGG TGG TGA TGA CCG TGA TCG) complementary to the sequence that encodes for the amino acid sequence ITVITTIG in KT3.2, and the forward primer AP1 form Clontech Marathon-Ready human brain cDNA Kit. A second nested amplification was performed using the oligonucleotide complementary to the area that encodes for the amino acid sequence LIVCTFTYL in the human KT3.2 cDNA (AGC AGG TAG GTG AAG GTG CAG ACG ATG AGG) and the internal forward primer AP2 form the Clontech Marathon-Ready human brain cDNA kit.

Cloning of rat KT3.2

Two degenerated oligonucleotides were designed against areas with significant sequence conservation in human mouse and rat TASK-1, human KT3.2, and Caenorhabditis elegans n2p38 (Vega-Saenz de Miera and Lin 1992). The forward primer (ATG AAG AGG CAR AAY RTN MGN AC) was designed using the sequences around the starting methionine (MKRQNV/IRT). The reverse primer (AAG GCA CCG ATN ACN GTN ARN CC) was designed to be complementary to the amino acid sequence (GLTVIGA) located in the fourth TMD of these channels. The latter sequence is also conserved in the human KT3.3 sequence. These primers were used for PCR amplification from cDNAs prepared from mRNA isolated from thalamus and cortex (Vega-Saenz de Miera and Lin 1992). The PCR conditions were identical to those used for the cloning of the human KT3.2 except that the annealing temperature was 48°C.

Cloning of human KT3.3

PCR was performed using the primers GCC ATG CGG AGG CCG AGC GT (forward) and CTA TGG TGG TGA TGA CCG TGA T (reverse), under the following PCR conditions: 35 cycles of 58°C for 1 min and 97°C for 1 min, using human genomic DNA as well human brain cDNAs as templates. The PCR products were separated in 2% agarose gels, and the amplified bands were subcloned as in the case of human KT3.2. The final sequence of KT3.3 was assembled by combining the sequences obtained from the expressed sequence tag (EST) clones (see RESULTS) and from the PCR band using an internal NotI site. The integrity of this construct was confirmed by double-strand sequencing.

mRNA isolation

Total RNA was prepared using the guanidinium-thiocyanate method (Chomczyznski and Sacchi 1987) from tissues obtained from adult (150-175 g) Sprague-Dawley rats. Poly(A) RNA was isolated with oligo(dT) cellulose columns as previously described (Rudy et al. 1988).

Northern and dot blot analysis

Human multi-tissue master dot blot (Clontech) was hybridized using Stratagene QuickHyb solution. The complete coding sequence of KT3.2 was labeled by random priming and used as a probe. The blot was hybridized for 1 h at 68°C with two washes at RT in 2× SSC 0.1% SDS and a final wash at 60°C in 0.1 SSC, 0.1% SDS for 30 min. The blot was placed at RT in a phosphoimager cartridge for 48 h. The values are reported as the values of the average signal measured by a circle that contains all the pixels in the dot minus the background, which was estimated by measuring an equal area free of signal but in the vicinity of the dot. The value was finally corrected for mRNA concentration applied to the blot and is reported as arbitrary digital counts per microgram of mRNA. Measurements of signal intensity were performed using Scion Imager.

The mRNAs isolated from rat tissues were electrophoresed in formaldehyde-denaturing gels and transferred to Duralon membranes (Stratagene). The membranes containing the bound mRNAs as well as a Clontech multi tissue rat Northern blot were hybridized with the first 719 nucleotides of the coding sequence of rat KT3.2. The probe was labeled with [32P]dCTP using the Boehringer random primers kit. The blots were hybridized in QuikHyb (Stratagene) containing 1× 106 cpm/ml (0.5-1.0 ng of DNA/ml) for 1 h at 68°C. They were then washed twice in 2× SSC, 0.1% SDS at room temperature for 15 min each, a final wash in 0.1× SSC, 0.3% SDS at 60°C for 30 min. The blots were exposed to X-ray film (X-OMAT AR Kodak) at -70°C for 1-2 days.

In situ hybridization

The in situ hybridization experiments were performed as described (Vega-Saenz de Miera et al. 1997), with the exception that a cDNA fragment made of the first 719 nucleotides of the coding sequence of rat KT3.2 was used as template for probe synthesis. Blast search using this sequence shows areas with <= 81% identity (105 bp) and 74% (474 bp) to rat Task-1 and 87% (41 bp), 81% (198 bp), and 76% (205 bp) to KT3.3 but not significant identities to other cDNAS. Specificity was further checked by hybridization of this probe against a blot containing synthetic RNA of rTASK-1 (Leonoudakis et al. 1998), rat KT3.2, and human KT3.3. A strong signal was with rat KT3.2, but no signal with rTASK or KT3.3 was obtained after 24 h (data not shown). Another evidence of specificity was obtained by the results of the Northern blot experiments in which the distribution as well as the size of the mRNA band do not mach to the ones described for rTASK (Leonoudakis et al. 1998).

Two-electrode voltage clamp of Xenopus laevis oocytes

The recombinant pCR2.1 plasmid containing KT3.2 was linearized by digestion with ScaI, and cRNA was transcribed in vitro using T7 RNA polymerase (Stratagene). Stage IV-V Xenopus oocytes were harvested, treated with collagenase (1 mg/ml), and micro-injected with 50 nl cRNA (~20 ng) (Iverson and Rudy 1990). All electrophysiology experiments were carried out at room temperature (22°C) 1-2 days following injection. Recordings were made in solutions containing ND96 (in mM): 1.8 CaCl2, 1 MgCl2, 96 NaCl, 2 KCl, and 5 HEPES, pH 7.5, or KD98, which is similar in composition to ND96 except that NaCl was replaced with KCl. Intermediate concentrations of potassium were obtained by mixing ND96 and KD98. Square voltage-clamp pulses were applied from a holding potential of -90 mV (ND96), -40 mV (KD14), -30 mV (KD26) -20 mV (KD50), or 0 mV (KD98) to test potentials ranging from -150 to +50 mV in 10-mV increments with a 3-s inter-pulse interval. Acquisition of data and subsequent analysis was performed using pClamp 6.0 (Axon Instruments) and Microsoft Excel.


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Human KT3.2

Screening of the GeneBank EST (expressed sequence tags) database using the Blast algorithm (Altschul et al. 1997) with the sequence of TASK-1 cDNA (Accession No. AF006823) as a probe yielded two EST sequences, AI690321 and AI739096, distinctly different from but related to TASK-1. The clones were incomplete at the 5' end and are partial sequences of KT3.3 (vide infra). Using the partial sequences of KT3.3 found in the EST database, a search in the high throughput genomic sequences database (HTGS) identified two human genomic sequences containing two new KT genes. One of the genes encodes a KT channel that had 58% identity and 72% similarity to the published human TASK-1 sequence (Accession No. AC007869) These values increase to 75% identity, 86% similarity if only the core region is used for comparison. We refer to this gene as KT3.2.

Analysis of the KT3.2 genomic sequence identified two exons that cover the full coding sequence of this channel. The exons are separated by an intron that is larger than 80 kb (Fig. 1A). Using the predicted coding sequence as a query in a search against the EST database revealed a sequence that contains the carboxy terminus of a KT3.2 transcript (AA349574) and served to confirm the position of the stop codon. A third exon, encoding the 3' UTR, was also identified. The first splice junction occurs at the first glycine of the GYG sequence in the first pore region. Oligonucleotide primers designed against sequences surrounding the predicted starting methionine and the stop codon of KT3.2 were used for PCR amplification with human brain cDNA (Clontech) as template. A band of 1,137 bp was observed with gel electrophoresis, and the cDNA was purified and subcloned. Three independent clones were identical in sequence and differed only at nucleotide 636 of the coding sequence from the putative sequence predicted by the analysis of the genomic sequence contained in AC007869 (a thymidine-to-cytosine substitution). This substitution does not alter the amino acid sequence and may represent a single nucleotide polymorphism (Fig. 1C). To confirm that the assigned starting methionine was the real starting methionine, five different RACE experiments using different primers were attempted. In each case, but in the last one, we obtain sequence related to KT3.2 that did not reach the starting methionine. In the last case, using the primers for the RACE experiments described in METHODS, we isolated 75 clones, 50 of which contained additional sequence related to KT3.2. From this experiment, we obtained 74 extra nucleotides without a stop codon in frame. The coding sequence found in the identified sequence predicts a protein of 374 aa with a molecular weight of 42,264 Da. Hydrophobicity analysis (Kyte and Doolittle 1982) predicts the presence of four transmembrane domains (Fig. 1B). The sequence contains two regions between the first and second and between the third and fourth TMDs having the canonical GYG/GFG sequence that is part of the pore of potassium channels (Heginbotham et al. 1994) (Fig. 1C). A potential N-glycosylation site (Fig. 1C, ) (Miletich and Broze 1990) is found in the linker between M1 and P1. Interestingly, as in TASK-1, this new protein does not have a cysteine in the extracellular M1-P1 that is found in all other mammalian KT channels and that has been suggested to be critical for channel assembly (Lesage et al. 1996c). The carboxy terminal sequence of the predicted protein contains three consensus sequences for phosphorylation by protein kinase C (Fig. 1C, ) (Woodgett et al. 1986) and one for PKA (Fig. 1C, black-triangle) (Feramisco et al. 1980; Glass et al. 1986).



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Fig. 1. Genomic organization, hydropathy profile, nucleotide and amino acid sequence of KT3.2 and KT3.3. A and D: genomic organization of KT3.2 and KT3.3 genes, respectively. , exons; , coding areas. Start, stop, and splice sites are indicated. Connecting introns are shown by lines and their sizes are indicated. Codon for AA 95 in KT3.2 and KT3.3 created by the junction of exons 1 and 2. B and E: hydrophobicity plot for KT3.2 and KT3.2, respectively (Kyte and Doolittle 1982). The areas of the transmembrane domain (TMD) as well as of the 2 pores are indicated. C and F: nucleotide and predicted amino acid sequence of KT3.2 and KT3.3, TMD, and pore regions are over lined. Consensus sites for: N-linked glycosylation (black-square), PKC (), and PKA (black-triangle) were identified as described in METHODS. These sequences have been deposited in GeneBank under Accession No. AF257080 for KT3.2 and AF257081 for KT3.3.

Human KT3.3

A Blast search using the partial EST sequences of KT3.3 described in the preceding text identified a new EST sequence, AI968607, which contains 140 bp in the 5' that do not match the previous ESTs. Translation of this extra sequence in one of the three reverse frames produces a 25 amino acid sequence with high degree of similarity to the sequences in the amino terminal of TASK-1 and KT3.2. We speculated that this could represent an inversion of the 5' sequence of the EST produced during library construction. PCR experiments using human genomic DNA or human brain cDNA with primers designed against the starting methionine predicted by the inverted 5' sequence and an internal primer based on the previous KT3.3 partial sequences produced a 283-bp product that was cloned and sequenced. The sequence of this product confirmed that the antisense sequence of AI968607 corresponds to the 5' sequence of KT3.3. Combination of the sequence obtained from the PCR experiment with the one obtained from EST AI690321 produced a 1,286-bp-long sequence with a 41-bp polyadenylation track. The open reading frame of this cDNA predicts a protein of 330 amino acids with a molecular weight of 36,130 Da (Fig. 1F).

Hydrophobicity analysis (Kyte and Doolittle 1982) predicts a protein containing four TMDs. The protein also includes two loops containing the canonical GYG/GFG signature of the K+ channel pore (Heginbotham et al. 1994), located between the first and second TMDs and the third and fourth TMDs (Fig. 1E). Two putative sites for protein kinase C (Fig. 1F, ) and one for protein kinase A (Fig. 1F, black-triangle) are also observed.

A Blast search of the HTGS database of GeneBank, using KT3.3 sequence as a probe identified a genomic sequence containing the KT3.3 gene (Accession No. AL118522). Analysis of KT3.3 sequence and the genomic clone AL118522 using Blast 2 sequences shows that the cDNA sequence is made by the participation of two different exons, separated by a 3,936-bp intron (Fig. 1D).

Expression of KT3.2 in Xenopus oocytes

Xenopus oocytes were injected with KT3.2 cRNA and studied after 18-48 h in a bath solution of ND96. These oocytes had a resting potential of -89 ± 2 mV (n = 15; P < 0.05), which was ~45 mV more negative than that of uninjected oocytes (-44 ± 2 mV; n = 7). This membrane potential was close to the K+ equilibrium potential (calculated to be -90 mV under our experimental conditions), suggesting that the expressed channels are selectively permeable to K+ and can play an important role in the generation of the resting potential.

Using two-electrode voltage-clamping techniques (in ND96 solution), mainly outward currents were observed. The currents activated almost instantaneously (Fig. 2A). These currents were absent in control water-injected oocytes (Fig. 3C). At high extracellular K+ concentrations (KD98), large inward currents were detected (Fig. 2B) although a weak outward rectification can be observed; (Fig. 2E). In this respect, KT3.2 differs from the closely related TASK-1, which shows a linear current-voltage relationship in high extracellular K+ concentrations (Duprat et al. 1997; Leonoudakis et al. 1998). The reversal potentials obtained from Fig. 2E, plotted as a function of the K+ concentration, yielded a linear relationship with a slope of 48 ± 1.3 mV per 10-fold change in K+ concentration (n = 7; Fig. 2F), a value that is close to the theoretical value of 58 mV, as predicted by the Nernst equation for a K+-selective channel. We were unable to detect any current (recorded in ND96 with a pH that ranges from pH 6.5 to 8.5) or shift in the resting potential in oocytes injected with KT3.3 cRNA.



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Fig. 2. Functional expression of KT3.2 in Xenopus oocytes. A: whole cell currents of KT3.2-injected oocytes recorded in ND96, from a holding potential of -90 mV to pulses from -150 to 50 mV in 10-mV increments. right-arrow, 0 current level. B: as in A but in KD98 and a holding potential of 0 mV. C and D: water injected oocytes in ND96 and KD98, respectively, under identical recording conditions as in A and B. E: plots of the currents recorded from oocytes expressing KT3.2 at the indicated K+ concentrations. F: reversal potential of KT3.2 currents as a function of [K+]o.



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Fig. 3. Effect of Ba2+ on KT3.2 currents. A: currents recorded in ND96 under similar conditions as in Fig. 2. B: same as A but in the presence of 3 mM Ba2+. C: plot of the current recorded with or without Ba2+ at 10 ms (triangle , no BA2+; open circle , with Ba2+) and after reaching the plateau (black-lozenge , no BA2+; , with Ba2+). Note than triangle  are on top of black-lozenge .

Modulation of KT3.2 currents by pharmacological agents and by pH

Currents produced by KT3.2 were relatively insensitive to extracellular application of TEA or 4-aminopyridine (4-AP; ~20% reduction in outward current was observed when all Na+ was replaced by TEA or 4-AP; data not shown). Moderate block was observed by extracellular quinidine (~20% block occurred with 100 µM and 80% block with 1 mM). Similar results were obtained using quinine (data not shown). Ba2+ (3 mM) added to the perfusion solution blocked the current. This blockade was released at potentials positive to the reversal potential in a time-dependent manner (Fig. 3B). After reaching steady state, inward currents are blocked proportionally more than outward currents (~80% block occurred at -150 mV, whereas only ~20% block occurred at +50 mV; Fig. 3C). This effect of Ba2+ is not due to changes in membrane surface charge because increasing Ca2+ to a final concentration of 4.8 mM or Mg2+ to a final concentration of 4 mM had no effect on KT3.2 currents (112 ± 6%, n = 3, for Ca2+ and 106 ± 6%, n = 3, for Mg2+).

KT3.2 currents were inhibited by the phorbol ester PMA, which is a potent PKC activator. A 50% inhibition was observed within 20 min of application of 10 nM PMA. An increase in PMA concentration to 100 nM produced similar results (Fig. 4). At 1 h after application, an 80% inhibition was observed (Fig. 4). The currents were not affected by incubation with the inactive phorbol ester 4alpha 4alpha-phorbol 12,13-didecanoate (PDD) (Fukushima et al. 1996) or in the absence of phorbol ester application (Fig. 4), suggesting that specific activation of PKC was responsible for the decrease in current produced by PMA. In contrast TASK-1 has been described to be insensitive to 100 nM PMA (Duprat et al. 1997).



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Fig. 4. Time course of the effect of phorbol esters on KT3.2 currents in Xenopus oocytes: average normalized currents recorded at 200 ms during a pulse to +50 mV from a holding potential of -90 mV. Results are the average of the recorded current normalized to the control current before any treatment. Error bars represent SD, n = 3.

KT3.2 and TASK-1 channels also differ in pH sensitivity. A characteristic of TASK-1 channels, from which its name was derived (TASK: two pore acid sensitive K+ channel), is their high sensitivity to pH changes around the physiological pH range, (current magnitude depends on pH with a pK of 7.29 ± 0.03 and is nearly completely blocked at pH 6.5) (Duprat et al. 1997; Leonoudakis et al. 1998). KT3.2 currents were also pH sensitive (Fig. 5), but the pH dependence is shifted toward more acid pH's by over 1 pH unit, such that KT3.2 currents were relatively insensitive to changes in the pH 7.0 to 8.0 range (Fig. 5). Furthermore, KT3.2 currents were not affected by application of dinitrophenol (1 mM for <= 20 min, data not shown), a treatment that presumably acidifies the cytosol and produces 50% reduction of TASK-1 currents after 6-min incubation (Leonoudakis et al. 1998).



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Fig. 5. Effect of pH on KT3.2 and TASK-1. A: current-voltage relationships determined at different pH's from KT3.2-injected Xenopus oocytes recorded during test potentials from -150 to 50 mV in 10-mV intervals from a holding potential of -90 mV. Please note than the pH 8.0 values are under the pH 7.4 curve. B: current-voltage relationships of TASK-1 at different pH (modified from Leonoudakis et al. 1998). C: percentage of the current remaining at different pH's. The currents were normalized to the current recorded at pH 8.5 (maximum current). The values of TASK-1 were obtained from Duprat et al. (1997).

Tissue distribution of human KT3.2

The pattern of KT3.2 tissue expression was studied by using multiple human tissues mRNA dot blots (Clontech). We found expression to occur mainly in neuronal tissue, particularly in cerebellum, although hybridization signals were also detected in samples of adrenal gland, kidney and lung mRNA (Fig. 6C). This expression pattern of KT3.2 is different from that of human TASK-1, which expresses more predominantly in placenta and pancreas followed by brain, lung, prostate, and heart (Duprat et al. 1997).



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Fig. 6. Pattern of tissue expression of human KT3.2. A: a multi-tissue master dot blot (Clontech) containing mRNAs from 50 different tissues was hybridized with the full-coding cDNA sequence of human KT3.2 under the conditions described in METHODS. The photograph shown was obtained after 5 days exposure at -70 with intensifier screens. B: dot blot index of the different human mRNAs. C: histogram of human KT3.2 concentration obtained after normalizing the signal recorded with the phosphoimager to 1 µg of mRNA using the values supplied by the membrane manufacturer.

Distribution of KT3.2 in rat tissues

To study the expression of the KT3.2 gene in rat, we generated species-specific probes. We used RT-PCR with degenerate oligonucleotides (see METHODS) with cortex and thalamus rat cDNAs as template. Two different sequences were isolated from these experiments. One was identical to the rat TASK-1 sequence (Leounoudakis et al. 1998), whereas the other encoded a sequence of 237 aa with 95% identity (99.6% similarity) to human KT3.2 (Fig. 7). This rat ortholog of KT3.2 was used as a probe for in-situ hybridization and Northern blot experiments.



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Fig. 7. Alignment of human and rat KT3.2. The partial sequence of rat KT3.2 was obtained by rtPCR from cDNAs prepared from brain cortex and thalamus using degenerated oligonucleotides and was deposited in GenBank with Accession No. AF257082. The predicted amino acid sequence of rat KT3.2 was aligned against the human KT3.2 sequence. Amino acid conservation is indicated by black background, TMD and pores are indicated by labeled overlines; the localization of the primers used to amplify the full sequence are indicated by arrows, and N-linked glicosylation sites by black-triangle.

Northern blot analysis revealed that the KT3.2 gene is expressed in the CNS but not in heart, lung, liver, kidney, intestine, or skeletal muscle. This pattern of expression is different from that of TASK-1, which in rat and mouse expresses mainly in heart and lung (Duprat et al. 1997; Leonoudakis et al. 1998) (Fig. 8). In situ hybridization experiments revealed a wide expression pattern in rat brain (Fig. 10, Table 1). The highest expression levels were found in the olfactory nuclei, piriform cortex, cerebellum, anterodorsal thalamic nucleus, pontine nucleus, dorsal raphe, and several nuclei in the medulla (Fig. 9). An intriguing expression pattern was found in hippocampus, with low levels of expression in the medial, anterior, and superior areas and higher levels in the lateral and ventral areas (Fig. 9, A, B, F, G vs. H and B). Diffuse expression was also observed throughout the cortex (Fig. 9, A-H and Table 1). In spinal cord, the channel is expressed in the anterior and posterior horns and more intensely in layer 9 of Rexed (Fig. 9M and Table 1).



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Fig. 8. Northern blot analysis of rat KT3.2. Top: X-ray autoradiogram of a rat multiple tissue mRNA blot hybridized with a probe made with the first 719 nucleotides of the coding sequence of rat KT3.2. The blot was exposed for 2 days at -70°C with intensifier screens. Bottom: photograph of the X-ray autoradiogram of the same blot following stripping and hybridization to a beta -actin probe. The film was exposed for 3 h at -70°C with intensifier screens. mRNA (2 µg) was applied to each lane except for the cerebellum lane in which 5 µg were applied. Cx, cortex; Cer, cerebellum; Hip, hippocampus; H, heart; Lg, lung; Lv, liver; K, kidney; I, intestine; M, muscle.


                              
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Table 1. Expression of KT3.2 in brain



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Fig. 9. Expression of KT 3.2 in the rat CNS. Autoradiograph of sagittal (A and B) and coronal sections at different levels (C-M) of rat CNS hybridized with random primed labeled 35S cDNA rat KT3.2 probe. aca, anterior commissure, anterior part; ac anterior comisure; Acg, anterior cingulate cortex; AD, anterodorsal thalamic nucleus; AHA anterior hypothalamic area, anterior part; Ahi, amygdalohippocampal area; AMB, anteromedial thalamic nucleus, ventral part; Amb, ambiguus nucleus; AO, anterior olfactory nucleus; AOD anterior olfactory nucleus, dorsal part; AOV anterior olfactory nucleus, ventral part; APT, anterior pretectal nucleus; Arc, arcuate nucleus; BL, basolateral amygdaloid nucleus; CA1 field CA1 of hippocampus; CA2 field CA2 of hippocampus; CA3 field CA3 of hippocampus; ml corpus callosum; CG, central gray; CGD, central gray, dorsal; Cli, caudal linear nucleus of the raphe; CnF, cuneiform nucleus; Cpu, caudate putamen; DCn deep cerebellar nuclei; Dco, dorsal cochlear nucleus; DG, dentate gyrus; DLG, dorsal lateral geniculate nucleus; DMC, dorsomedial hypothalamic nucleus, compact part; DR, dorsal raphe nucleus; Ent, entorrinal cortex; FrCx, frontal cortex;; Ge5, gelatinous layer of the caudal spinal trigeminal nucleus; Gr, cerebellar granular cell layer; IC, inferior colliculus; Int, interposed cerebellar nucleus, IO, inferior olive; IP, interpeduncular nucleus; LatC, lateral (dentate) cerebellar nucleus; LOT, nucleus of the lateral olfactory tract; LRt, lateral reticular nucleus; LSI, lateral septal nucleus, intermediate part; Lv, lateral ventricle; Lve, lateral vestibular nucleus; ME, median eminence; MG, medial geniculate nucleus; MGV, medial geniculate nucleus, ventral; Mi, mitral cell layer of the olfactory bulb; Mo5, motor trigeminal nucleus; Obgr, granular cell layer of the olfactory bulb; PCg, post cingulate cortex; Pir, piriform cortex; Pn, Pontine nucleus; PP, peripeduncular nucleus; PSCh, preoptic suprachiasmatic nucleus; SCh, supra chiasmatic nuclei; SNC, substantia nigra compacta; Sol, nucleus of the solitary tract; Sp5O, spinal trigeminal nucleus, oral part; SuG, superficial gray layer of the superior colliculus; TT, taenia tecta; VMH, ventromedial hypothalamic nucleus,; VP, ventral pallidum; 3, oculomotor nucleus; 3v, third ventricle; 7, facial nucleus; 9 spinal cord layer 9 of Rexed; 12, hypoglossal nucleus.

Chromosomal localization of KT3.2 and KT3.3

THE KT3.2 GENE IS LOCALIZED TO 8q24.1-3. A database search using the genomic sequence containing KT3.2 identified three sequence-tagged sites (STS), having Accession Nos. Z53064, Z53078, and G23634. These STSs correspond to markers D8S1741 and D8S1743, which are, respectively, localized in the Geneton frame at 161.51 Mbp (536.87 cR3000 in GeneMap'99 (http://www.ncbi.nlm.nih.gov/genemap/loc.cgi?ID=19131) and WI-14922, located at 698 cR from the top of chromosome 8 in the MIT-Whitehead frame (http://carbon.wi.mit.edu:8000/cgi-bin/contig/sts_info?sts=EST275360&database=release), and 536.87 cR3000 in GeneMap'99 (http://www.ncbi.nlm.nih.gov/genemap/loc.cgi?ID=19130).The fact that the three markers are located in the same general area strongly supports the localization of this gene to this area. Although these frames are not anchored to the cytogenetic map, it is very probable that, based on the data obtained from GeneMap'99, this gene is located at 8q24.1-3.

THE KT3.3 GENE IS LOCATED TO CHROMOSOME 20q13. Searching the STS database using the genomic sequence found in AL118522, we obtained four human STS Accession Nos., G18091, G18129, G07367, and AL031792. All of these sequences map to human chromosome 20. The sequence G07367 corresponds to the human STS marker WI-9624. The MIT map (http://carbon.wi.mit.edu:8000/cgi-bin/contig/sts_info/406) localized this marker to the chromosome 20 reference map at 286.01 cR from top of the Chr20 linkage group. In GeneMap 98 (http://www.ncbi.nlm.nih.gov/genemap98/map.cgi?MAP=GB4&BIN=581&MARK=WI-9624) it is localized at 56.6 cM (235.78 cR3000) from the top of chromosome 20. Although there is no strict correlation between these frames and the cytogenetic maps, it is very probable, based on the data contained in GeneMap98, that this gene is located at 20q12-13.1.


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We report here the identification of two new members of the emerging four TMDs or two-pore (KT) family of K+ channel pore forming subunits: KT3.2 and KT3.3. The KT family constitutes the largest family of K+-channel proteins in the C. elegans genome, with close to 40 KT genes identified (Wang et al. 1999). Sequence analysis suggest that about half of these genes can be grouped into subfamilies containing at least two members, while the remainder appear to be unique genes. Counting KT3.2 and KT3.3, a total of nine different KT genes have been identified in mammals. The identities in the core region (TMD1-TMD4) of mammalian KT proteins range from 25 to 75% (Table 2). As the number of identified genes grows, it becomes possible to identify groups within the same species having particularly large similarities that can be considered separate subfamilies (Fig. 10).


                              
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Table 2. Percent identities between core regions of KT channels



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Fig. 10. Phylogenetic tree of the K+ channel (KT) family. Alignment of the KT channels was produced with Clustal W (Thompson et al. 1994), and the tree was produced by the neighbor-joining method (Saitou and Nei 1987). The numbers represent the fractional differences of the sequence with respect to the node (i.e., 0.1 = 10% difference). TOK1 (yeast) was used as an outgroup gene. Except for n2p20 and n2p38 (Caenorhabditis elegans), all the remaining genes are human. The names in parentheses represent the corresponding KT channel nomenclature suggested in this paper.

With the cloning of the human KT3.2 and KT3.3 genes, a clear new subfamily with more than one member in the same species can be defined (Fig. 10). Human KT3.2 is 58% identical (72% similarity) to human TASK-1. In the core region (TMD1-TMD4), these values increase to 75% identity and 86% similarity, while KT3.3 shows 68% identity (81% similarity) to TASK-1. Thus TASK-1, KT3.2, and KT3.3 can be considered members of a subfamily that has at least three members in the same species (human). At least two members of this subfamily also exist in rat (rat KT3.2, this paper; rat TASK-1: Leonoudakis et al. 1998). Interestingly, the members of this subfamily are the only mammalian KT genes for which a clear ortholog can be found in the C. elegans genome, the n2p38 gene (Wang et al. 1999) (Table 2; Figs. 10 and 11).



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Fig. 11. Alignment of of the human K+ channels, KT3.2, KT3.3, and TASK-1, and of the C. elegans n2p38 and n2p20 genes. Level of amino acid similarities indicated by the darkness of background, TMD and pore regions are overlined. N-linked glycosylation site is indicated by black-triangle.

HTWIK (Lesage et al. 1996b) together with TWIK-2/TOSS (Chavez et al. 1999; Pountney et al. 1999) and TRAAK/KT4.1 (Fink et al. 1998; A. Ozaita and E. Vega-Saenz de Miera, unpublished results) together with TREK (Patel et al. 1999) may also define two additional KT subfamilies (Fig. 10), although the similarities within these subfamilies are less than in the TASK group (48.6 and 51.2% identity, respectively, in the core region; Table 2). Interestingly, another gene related to TREK, human KT2.2, obtained by analysis of genomic sequences (Accession No. AL122021), has 70% identities in the core region (Vega-Saenz de Miera, personal observation).

The emergence of several KT subfamilies led us to implement a nomenclature system following the model developed by Chandy et al. (1991) for the pore forming subunits of voltage-gated (Kv) K+ channels. A similar nomenclature has been applied to the inward rectifier family (Isomoto et al. 1997). Following these models, each independent KT gene was denominated KTx.y where the K stands for potassium and T stands for "two pore." The "x" refers to the subfamily number and the "y" to the member's number in that subfamily. Subfamilies and members within a subfamily are numbered according to their order of publication. Accordingly TASK-1 would be called KT3.1 because it was the first identified member of the third identified subfamily of KT genes. Hence, the newly discovered genes published in this manuscript were named KT3.2 and KT3.3. Although these genes could also be called TASK-2 and TASK-3, we should point out that the name TASK-2 has already been used in the literature for a gene that also expresses pH-sensitive K+ channels (TASK: two pore acid-sensitive K+ channel) but is evolutionary more distantly related (Fig. 10, Table 2).

In the case of the Kv family of K+-channel-forming subunits, the classification into subfamilies turned out to be quite useful since the subfamilies, in addition to reflecting closely related homologs, also appear to constitute functional groups. Kv subunits can form homomultimeric channels that are believed to be tetrameric (Coetzee et al. 1999). Subunits of the same subfamily can also interact and form functional heteromultimeric channels (reviewed in Coetzee et al. 1999). In the KT family, it is believed that the channels are formed by dimers of KT subunits (Lesage et al. 1996c). Whether KT subunits also form heteromeric channels and whether this is or not restricted to members of the same subfamilies remains to be investigated.

FUNCTIONAL ROLES OF KT CHANNELS. Several of the cloned KT subunits express K+ currents in heterologous expression systems, presumably by forming homodimers (Lesage et al. 1996). A few others, including TOSS (Pountney et al. 1999), KCNK6, KCNK7 (Salinas et al. 1999), and KT3.3 (this paper), do not express currents when introduced into heterologous expression systems.

Although there are some subtle differences, all KT subunits expressed to date form "leak"- or "open rectifier"-type K+ channels, i.e., the channels show little voltage- or time-dependent gating, and the currents show a dependence on extracellular K+ concentration close to that described by the Goldman-Hodgkin-Katz equation. Since they are open at voltages below the threshold for action potential generation, these channels are likely to influence the subthreshold behavior of neurons, including helping set the resting potential and the resting impedance of the cell. Such channels are of great interest because they can dictate the degree of responsiveness of a neuron to incoming synaptic inputs. KT channels might be one the main contributors to the K+ "leak" or resting current in neurons and one of the main determinants of the resting potential.

Of the nine KT genes known to date, seven are known to be expressed in brain, but not much is known about the cellular patterns of expression in this tissue. Nevertheless even from the little available data, it seems likely that many CNS neurons express more than one KT subunit. As shown here, KT3.2 is widely expressed within the CNS, and the same seems to be true for TRAAK/HKT4.1 (Fink et al. 1998; A. Ozaita and E. Vega-Saenz de Miera, unpublished data), and TREK (Fink et al. 1996). TASK-1 (Talley et al. 2000) and KT3.2 (this paper) appear to overlap in the cerebellar granule cell layer (the expression of TASK-1 in cerebellar granule cells has also been confirmed with specific antibodies) (Millar et al. 2000), the olfactory bulb, and several brain stem nuclei like the ambigual, motor trigeminal, facial, vagal, and hypoglossal nuclei.

Although many KT channels produce similar "leak" K+ currents, and they may have overlapping patterns of expression, they differ from each in their regulation by physiological factors. In this context, the strong sensitivity of TASK-1 channels to changes in extracellular pH in the physiological range and of TRAAK and TREK channels to arachidonic acid, are findings from heterologous expression studies of great interest. TRAAK and TREK-1 also respond to stress and might be mechanosensitive K+ channels (Maingret et al. 1999; Patel et al. 1998). This allows for multiple pathways of modulating the resting K+ conductance of neurons. Therefore the discovery of factors that modulate specific KT channels is crucial to understand their roles in neurons.

KT3.2 channels are notably different from those expressed by TASK-1 in their sensitivity to extracellular pH. The pH dependence of KT3.2 is shifted to more acidic pH's by 1 pH unit, with the result that KT3.2 is much less sensitive to changes in pH around physiological values than TASK-1 (Fig. 5). On the other hand, KT3.2 currents are strongly modulated by phorbol esters that activate PKC and do not affect TASK-1 channels (Duprat et al. 1997; Leonoudakis et al. 1998). The presence of both TASK-1 and KT3.2 channels in the same cell (as might be the case in cerebellar granule cells and in motoneurons) may allow for the independent modulation of the cell's resting K+ conductance by extracellular pH and by neurotransmitters that activate the PLC pathway.

CHROMOSOMAL MAPPING OF KT3.2 AND KT3.3. The KT3.2 gene was mapped to human chromosome 8q24.1-3. Several diseases have been mapped to this area, including benign neonatal febrile convulsion/KCNQ3 (Charlier et al. 1998; Lewis et al. 1993), childhood absence epilepsy (Fong et al. 1998), idiopathic general epilepsy (Zara et al. 1995), benign adult familial myoclonic epilepsy (Mikami et al. 1999), Lom syndrome, an autosomal recessive peripheral neuropathy with deafness (Kalaydjieva et al. 1996), and Meleda disease, a cardiac disease with cardiomegaly, electrocardiographic abnormalities, and episodes of ventricular tachycardia (Fischer et al. 1998). On the other hand, the KT3.3 gene was mapped to 20q13.1, an area in which BFNC, a rare kind of epilepsy, has been mapped (Singh et al. 1999). More interestingly, several tumors (Bossolasco et al. 1999; De Angelis et al. 1999; Palmedo et al. 1999; Tanner et al. 1995) show a duplication of this chromosomal area with multiple copies being present in the same cell, which may explain why all the KT3.3 ESTs found in the GenBank database are from tumor cells (AI690321, moderately differentiated endometrial adenocarcinoma; AW167075, uterus serous papillary carcinoma; AI739096, colon tumor; AI968607, pooled germ cell tumors; and AW073155 and AI097455, glioblastoma). A possible linkage of KT3.2 or KT3.3 to any of these diseases is worth further investigation.


    ACKNOWLEDGMENTS

This research was supported by National Institute of Neurological Disorders and Stroke Grants NS-30989 and NS-35215 to B. Rudy and by American Heart Association Grant-in-Aid 9951070T to E. Vega-Saenz de Miera.


    FOOTNOTES

Address for reprint requests: E. Vega-Saenz de Miera, Dept. of Physiology and Neuroscience, New York University School of Medicine, 550 First Ave., New York, NY 10016 (E-mail: vegae01{at}endeavor.med.nyu.edu).

Received 27 April 2000; accepted in final form 8 March 2001.


    NOTE ADDED IN PROOF

While this paper was under review, three papers reporting the sequence and functional properties of KT3.2 (TASK-3) in rat, guinea pig, and human were published (J Biol Chem 275: 8340-9347; J Biol Chem 275: 16650-16657; Mol Brain Res 82: 74-83).


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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society