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Plant Physiol. (1995) 109: 327-330 Rapid Communication Evidence That Plant K+ Channel Proteins Have Two Different Types of Subunits' Huixian Tang, Aurea C. Vasconcelos, and Cerald A. Berkowitz* Plant Science Department, Cook College (H.T., G.A.B.), and Bureau of Biological Research, Nelson Biological Laboratories (A.C.V.), Rutgers-The State University of New Jersey, New Érunswick, New Jersey 08903 Plant K+ channel proteins have been previously characterized as tetramers of membrane-spanning (Y subunit polypeptides. Recent studies have identified a 39-kD, hydrophilic polypeptide that is a structural component of purified animal K+ channel proteins. We have cloned and sequenced an Arabidopsis thaliana cDNA encoding a 38.4-kD polypeptide that has a sequence homologous to the animal K+ channel p subunit. Southern and northern analyses indicate the presence of a gene encoding this cDNA in the Arabidopsis genome and that its transcription product is present in Arabidopsis cells. To our knowledge, this is the first report to document the presence of K+ channel p subunits in plants. Current models of voltage-gated Kt channels in plants (Jan and Jan, 1994; Schroeder et al., 1994) present the holoenzyme as a tetramer of four similar or identical subunits. These a subunits have a molecular mass of approximately 80 kD and a molecular structure similar to the "Shaker" family of K' channel polypeptides found in a wide range of animal cells (Sussman, 1992). Analysis of the deduced amino acid sequences of the only two plant K+ channel gene products that have been cloned (KAT1 and AKT1) reveals their molecular structures to be those of a subunit polypeptides, with six membrane-spanning regions, a voltage sensor, and a selectivity filter/pore region, which is the ion conduction pathway (Anderson et al., 1992; Sentenac et al., 1992). Presumably, four of these a subunits co-assemble in plant cell membranes with their pore regions facing together and inward toward the central core of the protein, which lies on an axis perpendicular to the plane of the membrane (Jan and Jan, 1994; Schroeder et al., 1994). A critica1 step in the molecular characterization of these plant (i.e. Arabidopsis tkaliana) K+ channel a subunits is the expression of the mRNA encoding these polypeptides in heterologous systems such as Xenopus laevis oocytes. Only the translation product of the KATl cDNA has been studied in such a system (Schachtman et al., 1992). Results demonstrate that the translation product of the KATl gene is sufficient alone to confer Kt channel activity on the This material is based on work supported by the U.S. Department of Agriculture National Research Initiative Competitive Grants Program under award No. 92-01422-5586. * Corresponding author; e-mail [email protected]; fax 1-908-932-9441. target membrane. Patch/voltage-clamp analysis of Xenopus oocytes expressing the KATl gene product indicates that a functional, voltage-gated, inward-rectifying K+ channel can be formed (presumably) by self-assembly of four copies of the KATl polypeptide (Schachtman et al., 1992). Based primarily on this evidence and complementation of a K+ uptake-deficient mutant strain of yeast (Anderson et al., 1992), it has been thought that functional plant K+ channels are composed solely of these subunits. We present preliminary evidence in this report that native plant K' channel proteins likely are composed of a second, or /3, subunit. Evidence supporting this assertion, as presented in this report, is the cloning and sequencing of a cDNA from an A. thaliana expression library that encodes a polypeptide with a deduced amino acid sequence homologous to the sequences of a recently discovered class of polypeptides expressed in mammalian brain tissue. This class of polypeptides has been shown to be bound tightly to, and co-purify with, K+ channel a subunits isolated from native animal membranes (Scott et al., 1994). In addition to this biochemical evidence identifying these /3 subunit polypeptides as structural components of native K' channel proteins, functional studies with one member of the /3 subunit family have led to the initial identification of at least one biophysical role that they play in the proper functioning of the Kf channel holoenzyme (Rettig et al., 1994). To our knowledge, these K+ channel /3 subunit polypeptides have not been known to be present in plants. MATERIALS A N D M E T H O D S A GenBank search using the rat KJ31 cDNA sequence identified an Arabidopsis thaliana cDNA fragment (accession No. 218389) as a putative homolog. Oligonucleotide primers 1 and 2 (ATGGATCCACGCTGAGGTTTACGCT and GCGAATTCCACATCAACGTAATCC, respectively) corresponding to the 5' and 3' ends of the 218389 fragment, along with primers 3 and 4 (CATCTCTACCAAGATCTTCTGG and GAAGATCTTGGTAGAGATGACG, respectively), representing nested interna1 sequences, were synthesized. The DNA template used for primary PCR was 8 X 10' recombinant phage from a directionally cloned A ZAPII cDNA library constructed from A.thaliana Landsberg evecta inflorescences (obtained from the Ohio State University Arabidopsis Resource Center, Columbus, OH) as a template. 327 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 1995 American Society of Plant Biologists. All rights reserved. Tang et al. 328 Primary PCR was undertaken with primer 1 and the plasmid primer T7 and separately with primer 2 and the plasmid primer T3. PCR with primer 1 yielded a 1.1-kb DNA fragment corresponding to the 3' end of the target cDNA. PCR with primer 2 yielded a 0.45-kb fragment corresponding to the 5' end of the target cDNA. The conditions for a11 PCRs were: 5 p~ primers and 35 repeat cycles of 30 s at 94"C, 1 min at 55"C, and 1 min at 72"C, with a 10-min extension at 72°C for the last cycle prior to halting the reaction at 4°C. Two secondary PCRs were undertaken for reamplification. One secondary PCR used T7 primer, the nested primer 3, and the 1.1-kb product of the primary PCR (which used primer 1) as a template. The other secondary PCR used T3 primer, the nested primer 4, and the 0.45-kb product of the primary PCR (which used primer 2) as a template. The reamplified PCR fragments (0.3 kb for the 5' end and 1.0 kb for the 3' end of the original cDNA) were subcloned and sequenced. Sequence analysis led to the generation of primer 5 (TTGGATCCAACATGCAGTACAAGAATCTGG), corresponding to the 5' end of the cDNA, and primer 6 (TCGTCGACTTACCTATATGATTCAGGACG), corresponding to the 3' end of the cDNA. Primers 5 and 6 were used for PCR with the original cDNA library to generate a full-length DNA sequence from the target cDNA, which was then cloned and sequenced. A11 cloning reactions were done as follows. After size fractionation on 1% agarose gels, bands corresponding to PCR products were excised and purified with GeneClean I1 (Bio 101, La Jolla, CA). Purified DNA was blunt-end ligated into the EcoRV site of pBluescript KS(?) (Stratagene). Strain DHa5 F' competent cells were used as a host for the cloning vector. Dideoxy sequencing was performed under standard conditions using the United States Biochemical Sequenase Kit. Southern analysis from A . tkaliana (Columbia ecotype) used the method of Dellaporta et al. (1983) for DNA preparation. DNA was restriction enzyme digested with BamHI and electrophoresed on 0.8% agarose gels. DNA was denatured, neutralized, transferred to a nylon membrane, and cross-linked using standard procedures (Southern, 1975). The full-length cloned PCR product was 32P-labeled with a random-primer kit (Boehringer Mannheim). Prehybridization, hybridization, washing, and autoradiography followed standard protocols (Sambrook et al., 1989). RESULTS AND DISCUSSION Use of the (radiolabeled) mamba snake venom peptide dendrotoxin, a potent K+ channel blocker, led to the firstever purification of a K+ channel protein (Parcej and Dolly, 1989). Chromatographic purification of the dendrotoxinreceptor K+ channel protein from bovine cerebral cortex synaptic plasma membranes has identified a 39-kD polypeptide as a component of the holoenzyme (Parcej et al., 1992). A 78-kD polypeptide (the a subunit) was also found to be a component of this K+ channel protein in this work. N-terminal sequencing of the larger of the co-purifying polypeptides (Scott et al., 1990) confirmed that it was a K C channel a subunit; the sequenced portion of this polypeptide was identical with the N-terminal sequence Plant Physiol. Vol. 109, 1995 deduced from a cloned cDNA encoding a known Kt channel a subunit. Further evidence identifying the 39-kD polypeptide as a structural component of K+ channel proteins is as follows. Cross-linking studies (Muniz et al., 1990) demonstrated that dendrotoxin bound only to the larger polypeptide (i.e. the a subunit). However, the 39-kD polypeptide was retained along with the a subunit on a dendrotoxin affinity column (Scott et al., 1990). Monoclonal antibodies raised against the a subunit were found not to immunoreact directly with the 39-kD polypeptide but were able to coimmunoprecipitate the smaller polypeptide along with the a subunit (Muniz et al., 1992). Although the 39-kD polypeptide was found not to be disulfide linked or covalently bound to the a subunit, the association between the two subunits could not be broken by exposure to a high concentration of salt (Dolly et al., 1994). Finally, hydrodynamic studies (using Suc gradients) of the dendrotoxmbinding complex purified from bovine cerebral cortex plasmalemma identified the K+ channel protein as composed of four of the 39-kD polypeptides along with four a subunits (Parcej et al., 1992). Based on this extensive evidence, it was concluded that the bovine brain 39-kD polypeptide was a K' channel p subunit. It is not entirely clear at present what functional role the p subunit from bovine brain plays in the K+ channel protein. A full-length cDNA encoding the bovine brain /3 subunit (KJ2) was recently cloned (Scott et al., 1994) and used to screen a rat brain cDNA library (Rettig et al., 1994). Two clones showing sequence homology were identified: rat K,Pl and rat K$2. One of the rat cDNAs, KvP2, encodes a deduced amino acid sequence sharing 99% identity with the 367-amino acid bovine K$2 sequence. The other rat clone, K,Pl, encodes a longer polypeptide (401 amino acids). Figure 1 shows the deduced sequences of rat KJ1 and bovine KvP2. The first N-terminal 72 amino acids of rat KVP1do not align with the N termini of bovine KvP2 (Fig. 1) or rat K,@2 (not shown). The rest of the K$1 sequence shares 85%amino acid identity with these other P subunits. It should be noted that Dolly et al. (1994) claimed that the mammalian K t channel P subunits are not related (by sequence comparison) with any other known proteins. We have identified a cDNA from an A . tkaliana expression library that appears to be a plant homolog to the mammalian brain K' channel /3 subunits. The deduced amino acid sequence of the plant cDNA encoding the K' channel Arabidopsis Beta subunit (KAB1) is shown aligned with bovine K,P2 and rat K,Pl in Figure 1. The KABl cDNA encodes a polypeptide with 328 amino acids. KABl polypeptide shares 49% sequence identity and 70% similarity (i.e. including conservative substitutions) with bovine K,P2 (Fig. 1). The nucleotide sequence of the cDNA encoding KABl is shown in Figure 2. The full-length KABl cDNA contains 1394 bp. The 987-bp open reading frame of this cDNA encodes a 38.4-kD polypeptide. The cDNA has a polyadenylation signal sequence (bp 1359-1364), an inframe stop codon upstream from the start codon (bp -35 to -33), and a Kozak consensus sequence at the correct position relative to the ATG start codon (Fig. 2). Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 1995 American Society of Plant Biologists. All rights reserved. Plant K + Channels Have Two Subunits ratKvBl bovKvj)2 MQVSIACTEHNLKSRNGEDRLLSKQSSTAPNWNAARAKFRTVAIIARSL ——————— _ ———————————— ——————————— MYPESTTGSPARLSLR KABl ratKvpl bovKvp2 ratKvpl bovKvp2 KABl 50 16 0 329 1 GGCACGAGAA GAGAGAGAGA GCGATAGTGA GA1TTAGATC AACAGATTTG 51 AATCGATTTCTGAAAACATGCAGTACAAGAATCTGGGGAAATCGGGTTTA AAAGTGAGCA CGCTCTCGTT CGGAGCGTGG GTTACGTTCG GGAACCAGCT QTPTPQHHISLKESTAKQTGMKYRNLGKSGLRVSCLGLGTWVTFGGQISD QTGSPGMIYSTRYGSPKRQLQFYRNLGKSGLRVSCLGLGTWVTFGGQITD ——————————— _ —————— MQYKNLGKSGLKVSTLSFGAWVTFGNQLDV 100 101 66 30 151 CGATGTGAAA GAAGCGAAAT CGATTCTTCA GTGTTGTCGT GATCATGGAG EVAERLMTIAYESGVNLFDTAEVYAAGKAEVILGSIIKKKGWRRSSLVIT EMAEHLMTLAYDNGINLFDTAEVYAAGKAEWLGNIIKKKGWRRSSLVIT KEAKSILQCCRDHGVNFFDNAEVYANGRAEEIMGQAIRELGWRRSDIVIS 150 116 80 201 TCAATTTCTT CGATAACGCT GAGGTTTACG CTAATGGTCG CGCTGAGGAG 251 ATTATGGGTC AAGCGATTCG TGAACTGGGT TGGCGTCGAT CCGATATCGT 0 ratKvBl bovKvp2 KABl TKLYWGGKAETERGLSRKHIIEGLKGSLQRLQLEYVDWFANRPDSNTPM TKIFWGGKAETERGLSRKHIIEGLKASLERLQLEYVDWFANRPDPNTPM TKIFWGGPGPNDKGLSRKHIVEGTKASLKRLDMDYVDVLYCHRPDASTPI 200 166 130 EEIVRAMTHVINQGMAMYWGTSRWSAMEIMEAYSVARQFNMIPPVCEQAE EETVRAMTHVINQGMAMYWGTSRWSSMEIMEAYSVARQFNLIPPICEQAE EEAVRAMNYVIDKGWAFYWGISEWSAQQITEAWGAADRLDLVGPIVEQPE 250 216 180 YHLFQREKVEVQLPELYHKIGVGAMTWSPLACGIISGKYGNGV-PESSRA YHMFQREKVEVQLPELFHKIGVGAMTWSPLACGIVSGKYDSGI-PPYSRA YNMFARHKVETEFLPLYTNHGIGLTTWSPLASGVLTGKYNKGAIPSDSRF O 299 265 230 SLKCYQWLKERIVSEEGRKQQNKLKDLSPIAERLGCTLPQLAVAWCLRNE SLKGYQWLKDKILSEEGRRQQAKLKELQAIAERLGCTLPQLAIAWCLRNE ALENYKNLANRSLVDDVLR- - -KVSGLKPIAGELGVTLAQLAIAWCASNP o 349 315 277 GVSSVLLGSSTPEQLIEMLGAIQVLPKMTSHWNEIDNILRNKPYSKKDY GVSSVLLGASNAEQLMENIGAIQVLPKLSSSIVHEIDSILGNKPYSKKDY NVSSVITGATRGSQIQENMKAVDVIPLLTPIVLDKIEQVIQSKPKRPESY RS RS R- o ratKvBl bovKvB2 KABl ratKvpl bovKvp2 KABl ratKvpl bovKvB2 KABl • O ratKvpl bovKvp2 KABl • ratKvBl bovKvp2 KABl o o O 301 CATCTCTACC AAGATCTTCT GGGGTGGTCC TGGTCCTAAC GATAAGGGTT 351 TATCTAGGAA ACATATCGTT GAAGGCACTA AAGCTTCTCT CAAACGACTT 401 GATATGGATT ACGTTGATGT GCTCTATTGC CACAGGCCGG ATGCTTCAAC 451 TCCTATCGAA GAGGCTGTGA GGGCGATGAA CTACGTGATT GATAAGGGTT 501 GGGCCTTCTA TTGGGGAATC AGTGAATGGT CAGCTCAACA AATTACGGAG 551 GCATGGGGAG CTGCTGACCG GTTGGATTTG GTTGGTCCAA TTGTCGAACA 601 GCCAGAATAC AACATGTTCG CTAGGCACAA AGTTGAGACA GAGTTTCTTC 651 CTCTGTACAC CAACCATGGT ATAGGTCTCA CTACCTGGAG CCCACTTGCA 701 TCTGGTGTGC TCACTGGTAA ATACAACAAG GGAGCTATTC CCTCAGACAG 399 365 327 751 CCGATTTGCA TTGGAGAACT ACAAAAACCT TGCCAATAGA TCACTTGTGG 801 ATGACGTGCT GAGGAAAGTT AGCGGTCTCA AACCCATTGC AGGTGAGCTA 401 367 328 851 GGTGTAACCT TGGCTCAGCT TGCAATCGCA TGGTGTGCTT CAAATCCTAA Figure 1. Deduced polypeptide sequences (single amino acid code) of cloned K + channel ft subunits from rat (ratK v /31), bovine (bovKj32), and A. thaliana (KABl). Potential phosphorylation and N-glycosylation sites in the KABl sequence are identified by open and filled circles, respectively. Shaded areas identify sections of the KABl sequence that share identity with bovine Kv/32 or bovine Kv/32 and rat KJ31. 901 TGTGTCATCT GTTATCACTG GTGCCACAAG GGGGTCACAG ATTCAAGAAA 951 ATATGAAAGC TGTTGATGTG ATCCCATTGT TGACCCCTAT TGTTCTGGAC 1001 AAGATTGAGC AAGTGATACA GAGCAAACCA AAACGTCCTG AATCATATAG 1051 GTAAAACCAA CATCCAAGAT CTCTCTTCCC TATTCAATCG TTTACAAAAG 1101 AGTGTTGCAG GAAAAAGAAA ACATTAGAAG AAGCTCTGTG ATGTATGTTG 1161 TTGGATGTTG TCTCGTTTTC GCTTTGTTTG TTCTCTTTAG CAGCTTATCA 1201 TTTTTAAGAC TCAGACAGAG AGAAAGAGAG ACTAATGTTT TTTTTTTAGT 1251 TTTTCTTGTT TCATCATTTA AAAAACGGTC TTATTTGTTA CTTGTTAGTG 1301 CAGCTTAAAG TTTGGTTCTT GTAGTTTGCC ATGTCATGAC GTCAATATAT 1351 TGAATAGCTA ATAAAACAAT TCTGGTTAAA AAAAAAAAAA AAAA Analysis of the deduced KABl amino acid sequence leads to some preliminary structural information about the Figure 2. Oligonucleotide sequence of KABl cDNA. The start and KABl gene product. Hydropathy analysis of mammalian stop codons and the polyadenylation signal sequence are shaded. brain K + channel /3 subunits (Rettig et al., 1994; Scott et al., 1994) indicates that they are hydrophilic polypeptides with the presence of a coding sequence(s) homologous to KABl no membrane-spanning regions. Mammalian /3 subunits in the Arabidopsis genome (Fig. 4). Northern analysis, were also found in these studies not to contain any potenagain with KABl as a probe, of poly(A + ) RNA isolated tial N-glycosylation sites, but they had numerous phosfrom Arabidopsis seedlings indicated the presence of phorylation sites. Accordingly, in vitro studies demonKABl-homologous mRNA (data not shown). strated that the bovine /3 subunit could be phosphorylated + The identification of a K channel /3 subunit in plants in the presence of protein kinase A, and the native /3 most certainly raises more questions than are answered by subunit was found not to be glycosylated in vivo (Scott et the work presented in this report. The primary issue of al., 1994). Because of their hydrophilic nature, their capacfunctionality is left unresolved. However, recent studies ity to be phosphorylated, and the absence of glycosylation (Rettig et al., 1994) with rat Kv/31 have led to the exciting or membrane-spanning regions, mammalian /3 subunits are hypothesis that )3 subunit polypeptides may be acting as currently thought to reside in the cytoplasm, where they the "inactivation gate" of the K + channel protein. In this interact with the cytoplasmic portion of the membranetraversing a subunits. Even though the KABl gene product shares substantial homology with mammalian /3 subunits 20' (Fig. 1), there are some significant differences. KABl has 8 phosphorylation sites (Fig. 1) but retains only 3 of the 13 10' (Rettig et al., 1994) mammalian |3 subunit phosphorylation sites. Hydropathy analysis (Fig. 3) of KABl indicates an 0' overall hydrophilic nature. However, amino acids 261 to 287 contain no charged side chains. This string of 27 amino -10' acids in the KABl sequence could, therefore, be a membrane-spanning section of the polypeptide. It is intriguing -20" ' ' ' • ' ' ' • ' I " ' ' " ' ' ' I " " " ' ' ' I " " ' " " I " ' ' " ' ' ' I " ' ' that KABl has two potential glycosylation sites (Fig. 1) 1 60 120 180 240 300 near this putative membrane-spanning region. Figure 3. Hydropathy plot (positive values are hydrophilic) of deFurther work suggested that the KABl gene product is a duced KABl amino acid sequence. The method of Kyte and Doolitlikely constituent of plant (at least Arabidopsis) cells. tle (1982), with a five-amino acid interval size, was used for this Southern analysis with radiolabeled KABl cDNA indicated analysis. Downloaded from on June 18, 2017 - Published by www.plantphysiol.org ^•ul'W i \jw ji n i !• Copyright © 1995 American Society of Plant Biologists. All rights reserved. Tang et al. 330 Plant Physiol. Vol. 109, 1995 and KAB1 act in vivo to affect gating of K+ channel a subunits. However, even though the role KAB1 plays as part of K+ channels in vivo remains unresolved, it is unequivocally homologous to bovine Kvj32 (Fig. 1); bovine Kv/32 has been thoroughly documented to be a structural component of native animal K+ channel proteins. 4.2Kb — 2.1Kb— «• Figure 4. Southern blot of A. thaliana genomic DNA digested with BamHI (B1) and probed with the KAB1 cDNA. Received March 8, 1995; accepted June 5, 1995. Copyright Clearance Center: 0032-0889/95/109/0327/04. The GenBank accession number for the sequence reported in this article is L40948. LITERATURE CITED Anderson JA, Huprikar SS, Kochian LV, Lucas WJ, Gaber RF (1992) Functional expression of a probable Arabidopsis thaliana potassium channel in S. cerevisiae. Proc Natl Acad Sci USA 89: work, co-expression of Kv/31 with the K+ channel a subunit 3736-3740 RCK1 in Xenopus oocytes altered the gating characteristics Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA miniprepaof RCK1-induced currents. RCK1 had been previously ration: version II. Plant Mol Biol Rep 1: 19-21 thought to be a delayed-rectifier type of voltage-gated K+ Dolly JO, Rettig J, Scott VES, Parcej DN, Wittka R, Sewing S, Pongs O (1994) Oligomeric and subunit structures of neuronal channel. Co-expression of Kvj31 with RCK1 transformed voltage-sensitive K+ channels. Structure and regulation of catthe induced currents into those representative of another ion channels. Biochem Soc Trans 22: 473^478 subclass of voltage-gated K+ channels, i.e. fast-inactivating Jan LY, Jan YN (1994) Potassium channels and their evolving A type. These results led to the hypothesis that /3 subunit gates. Nature 371: 119-122 polypeptides (at least Kv/31) act as a "ball-and-chain" Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157: 105-132 mechanism, with the "ball" physically occluding the ion Muniz ZM, Diniz CR, Dolly JO (1990) Characterisation of binding conduction pathway formed by the pore regions of four a sites for 8-dendrotoxin in guinea-pig synaptosomes: relationship subunits. As shown by Rettig et al. (1994), delayed-rectifier to acceptors for the K+ channel probe a-dendrotoxin. J Neurocurrents expressed by cloned a subunits do not deactivate chem 54: 343-346 Muniz ZM, Parcej DN, Dolly JO (1992) Biochemistry 31: 12297(the channel stays open) for relatively long periods (20% 12303 current decay after 10 s for RCK1). Co-expression of Kvj31 Parcej DN, Dolly JO (1989) Dendrotoxin acceptor from bovine results in a 5-ms half-time for deactivation of RCK1. Rettig synaptic plasma membranes. Binding properties, purification et al. (1994) noted that numerous studies of native memand subunit composition of a putative constituent of certain voltage-activated K+ channels. Biochem J 257: 899-903 branes have documented the presence of A-type voltage+ + Parcej DN, Scott VES, Dolly JO (1992) Oligomeric properties of gated K channels but that the majority of cloned K a-dendrotoxin-sensitive potassium ion channels purified from channel a subunits display noninactivating currents upon bovine brain. Biochemistry 31: 11084-11088 expression. They postulated that the "standard" configuRettig J, Helnemann SH, Wunder F, Lorra C, Parcej DN, Dolly ration of A-type channels in vivo includes /3 subunits. It is JO, Pongs O (1994) Inactivation properties of voltage-gated K+ channels altered by presence of /3-subunit. Nature 369: 289-294 intriguing for us to note that the only plant a subunit Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A cDNA expressed in Xenopus oocytes displayed delayedLaboratory Manual. Cold Spring Harbor Press, Cold Spring rectifier, noninactivating currents. In a recent review, Harbor, NY, pp 9.31-9.58 + Schroeder et al. (1994) alluded to the notion that K chanSchachtman DP, Schroeder JI, Lucas WJ, Anderson JA, Gaber RF (1992) Expression of an inward-rectifying potassium channel by nels in plants serve very different functions than in animal the Arabidopsis KAT1 cDNA. Science 258: 1654-1658 cells, i.e. in plants they should be designed for long-term Schauf CL, Wilson KJ (1987) Properties of single K+ and Cl~ K+ influx. They went on to postulate that it is sensible that channels in Asclepias tuberosa protoplasts. Plant Physiol 85: plant K+ channels lack an inactivation mechanism. How413-418 + ever, patch/clamp studies of native plant K channels do Schroeder JI, Ward JM, Gassman W (1994) Perspectives on the physiology and structure of inward-rectifying K+ channels in show voltage-dependent inactivation (Schauf and Wilson, + higher plants: biophysical implications for K + uptake. Annu Rev 1987). Documentation of the presence of a K channel ft Biophys Biomol Struct 23: 441-471 subunit in plant cells as presented in this report raises the Scott VES, Parcej DN, Keen JN, Findlay JBC, Dolly JO (1990) possibility that more research may be required to resolve a-Dendrotoxin acceptor from bovine brain is a K+ channel protein. J Biol Chem 265: 20094-20097 the overall structure and inactivation properties of native Scott VES, Rettig J, Parcej DN, Keen JN, Findaly JBC, Pongs O plant K + channel proteins. (1994) Primary structure of a ft subunit of a-dendrotoxin-sensiFurther work presented by Rettig et al. (1994) demontive K+ channels from bovine brain. Proc Natl Acad Sci USA 91: strated that the extreme N-terminal portion of Kvj31 was 1637-1641 critical for a subunit inactivation; this section of Kv|31 is Sentenac H, Bonneaud N, Minet M, Lacroute F, Salmon J-M, Gaynard F, Grignon C (1992) Cloning and expression in yeast of absent from rat Kv|32, bovine Kv/32, and the KAB1 sequence a plant potassium ion transport system. Science 256: 683-685 presented here. Co-expression of rat Kv/32 did not alter Southern EM (1975) Detection of specific sequences among DNA inactivation profiles of K+ channel a subunits (Rettig et al., fragments separated by gel electrophoresis. J Mol Biol 98: 1994). Our KAB1 sequence is similar to bovine and rat 503-517 Downloaded from on K June 18, 2017 - Published www.plantphysiol.org Sussman MR by (1992) Shaking Arabidopsis thaliana. Science 256: 619 Kvj32. It is unclear, therefore, whether rat and bovine v/32 Copyright © 1995 American Society of Plant Biologists. All rights reserved.