evidence that plant k+ channel proteins have - plant physiology

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

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 in- dicate the presence of a gene encoding this cDNA in the Arabidop- sis genome and that its transcription product is present in Arabi- dopsis 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 approx- imately 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 stud- ied 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. Depart- ment 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.

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New Jersey, New Érunswick, New Jersey 08903

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 cop- ies 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 METHODS

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 (CATCTCTACCAAGATCT- TCTGG and GAAGATCTTGGTAGAGATGACG, respectively), representing nested interna1 sequences, were syn- thesized. 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 Univers- ity Arabidopsis Resource Center, Columbus, OH) as a template.

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328 Tang et al. Plant Physiol. Vol. 109, 1995

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 (TTGGATCCAACATGCAGTA- CAAGAATCTGG), corresponding to the 5' end of the cDNA, and primer 6 (TCGTCGACTTACCTATATGAT- TCAGGACG), 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 prep- aration. DNA was restriction enzyme digested with BamHI and electrophoresed on 0.8% agarose gels. DNA was de- natured, 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). Prehy- bridization, 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 first- ever purification of a K+ channel protein (Parcej and Dolly, 1989). Chromatographic purification of the dendrotoxin- receptor 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-puri- fying polypeptides (Scott et al., 1990) confirmed that it was a KC channel a subunit; the sequenced portion of this polypeptide was identical with the N-terminal sequence

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 re- tained 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 co- immunoprecipitate 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 co- valently 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, hydrody- namic studies (using Suc gradients) of the dendrotoxm- binding complex purified from bovine cerebral cortex plas- malemma 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 KVP1 do 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% simi- larity (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 in- frame stop codon upstream from the start codon (bp -35 to -33), and a Kozak consensus sequence at the correct posi- tion relative to the ATG start codon (Fig. 2).

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Plant K+ Channels Have Two Subunits 329

ratKvBlbovKvj)2KABl

ratKvplbovKvp2

ratKvplbovKvp2KABl

ratKvBlbovKvp2KABl

ratKvBlbovKvB2KABl

ratKvplbovKvp2KABl

ratKvplbovKvB2KABl

ratKvplbovKvp2KABl

ratKvBlbovKvp2KABl

MQVSIACTEHNLKSRNGEDRLLSKQSSTAPNWNAARAKFRTVAIIARSL——————— _ ———————————— ——————————— MYPESTTGSPARLSLR

QTPTPQHHISLKESTAKQTGMKYRNLGKSGLRVSCLGLGTWVTFGGQISDQTGSPGMIYSTRYGSPKRQLQFYRNLGKSGLRVSCLGLGTWVTFGGQITD——————————— _ —————— MQYKNLGKSGLKVSTLSFGAWVTFGNQLDV

EVAERLMTIAYESGVNLFDTAEVYAAGKAEVILGSIIKKKGWRRSSLVITEMAEHLMTLAYDNGINLFDTAEVYAAGKAEWLGNIIKKKGWRRSSLVITKEAKSILQCCRDHGVNFFDNAEVYANGRAEEIMGQAIRELGWRRSDIVIS

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YHLFQREKVEVQLPELYHKIGVGAMTWSPLACGIISGKYGNGV-PESSRAYHMFQREKVEVQLPELFHKIGVGAMTWSPLACGIVSGKYDSGI-PPYSRAYNMFARHKVETEFLPLYTNHGIGLTTWSPLASGVLTGKYNKGAIPSDSRF

OSLKCYQWLKERIVSEEGRKQQNKLKDLSPIAERLGCTLPQLAVAWCLRNESLKGYQWLKDKILSEEGRRQQAKLKELQAIAERLGCTLPQLAIAWCLRNEALENYKNLANRSLVDDVLR- - -KVSGLKPIAGELGVTLAQLAIAWCASNP

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Figure 1. Deduced polypeptide sequences (single amino acid code)of cloned K+ channel ft subunits from rat (ratKv/31), bovine(bovKj32), and A. thaliana (KABl). Potential phosphorylation andN-glycosylation sites in the KABl sequence are identified by openand filled circles, respectively. Shaded areas identify sections of theKABl sequence that share identity with bovine Kv/32 or bovine Kv/32and rat KJ31.

Analysis of the deduced KABl amino acid sequenceleads to some preliminary structural information about theKABl gene product. Hydropathy analysis of mammalianbrain K+ channel /3 subunits (Rettig et al., 1994; Scott et al.,1994) indicates that they are hydrophilic polypeptides withno membrane-spanning regions. Mammalian /3 subunitswere also found in these studies not to contain any poten-tial N-glycosylation sites, but they had numerous phos-phorylation sites. Accordingly, in vitro studies demon-strated that the bovine /3 subunit could be phosphorylatedin the presence of protein kinase A, and the native /3subunit was found not to be glycosylated in vivo (Scott etal., 1994). Because of their hydrophilic nature, their capac-ity to be phosphorylated, and the absence of glycosylationor membrane-spanning regions, mammalian /3 subunits arecurrently thought to reside in the cytoplasm, where theyinteract with the cytoplasmic portion of the membrane-traversing a subunits. Even though the KABl gene productshares substantial homology with mammalian /3 subunits(Fig. 1), there are some significant differences. KABl has 8phosphorylation sites (Fig. 1) but retains only 3 of the 13(Rettig et al., 1994) mammalian |3 subunit phosphorylationsites. Hydropathy analysis (Fig. 3) of KABl indicates anoverall hydrophilic nature. However, amino acids 261 to287 contain no charged side chains. This string of 27 aminoacids in the KABl sequence could, therefore, be a mem-brane-spanning section of the polypeptide. It is intriguingthat KABl has two potential glycosylation sites (Fig. 1)near this putative membrane-spanning region.

Further work suggested that the KABl gene product is alikely constituent of plant (at least Arabidopsis) cells.Southern analysis with radiolabeled KABl cDNA indicated

1 GGCACGAGAA GAGAGAGAGA GCGATAGTGA GA1TTAGATC AACAGATTTG

51 AATCGATTTCTGAAAACATGCAGTACAAGAATCTGGGGAAATCGGGTTTA

101 AAAGTGAGCA CGCTCTCGTT CGGAGCGTGG GTTACGTTCG GGAACCAGCT

151 CGATGTGAAA GAAGCGAAAT CGATTCTTCA GTGTTGTCGT GATCATGGAG

201 TCAATTTCTT CGATAACGCT GAGGTTTACG CTAATGGTCG CGCTGAGGAG

251 ATTATGGGTC AAGCGATTCG TGAACTGGGT TGGCGTCGAT CCGATATCGT

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

751 CCGATTTGCA TTGGAGAACT ACAAAAACCT TGCCAATAGA TCACTTGTGG

801 ATGACGTGCT GAGGAAAGTT AGCGGTCTCA AACCCATTGC AGGTGAGCTA

851 GGTGTAACCT TGGCTCAGCT TGCAATCGCA TGGTGTGCTT CAAATCCTAA

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

Figure 2. Oligonucleotide sequence of KABl cDNA. The start andstop codons and the polyadenylation signal sequence are shaded.

the presence of a coding sequence(s) homologous to KABlin the Arabidopsis genome (Fig. 4). Northern analysis,again with KABl as a probe, of poly(A+) RNA isolatedfrom Arabidopsis seedlings indicated the presence ofKABl-homologous mRNA (data not shown).

The identification of a K+ channel /3 subunit in plantsmost certainly raises more questions than are answered bythe work presented in this report. The primary issue offunctionality is left unresolved. However, recent studies(Rettig et al., 1994) with rat Kv/31 have led to the excitinghypothesis that )3 subunit polypeptides may be acting asthe "inactivation gate" of the K+ channel protein. In this

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Figure 3. Hydropathy plot (positive values are hydrophilic) of de-duced KABl amino acid sequence. The method of Kyte and Doolit-tle (1982), with a five-amino acid interval size, was used for thisanalysis.

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330 Tang et al. Plant Physiol. Vol. 109, 1995

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Figure 4. Southern blot of A. thaliana genomic DNA digested withBamHI (B1) and probed with the KAB1 cDNA.

work, co-expression of Kv/31 with the K+ channel a subunitRCK1 in Xenopus oocytes altered the gating characteristicsof RCK1-induced currents. RCK1 had been previouslythought to be a delayed-rectifier type of voltage-gated K+

channel. Co-expression of Kvj31 with RCK1 transformedthe induced currents into those representative of anothersubclass of voltage-gated K+ channels, i.e. fast-inactivatingA type. These results led to the hypothesis that /3 subunitpolypeptides (at least Kv/31) act as a "ball-and-chain"mechanism, with the "ball" physically occluding the ionconduction pathway formed by the pore regions of four asubunits. As shown by Rettig et al. (1994), delayed-rectifiercurrents expressed by cloned a subunits do not deactivate(the channel stays open) for relatively long periods (20%current decay after 10 s for RCK1). Co-expression of Kvj31results in a 5-ms half-time for deactivation of RCK1. Rettiget al. (1994) noted that numerous studies of native mem-branes have documented the presence of A-type voltage-gated K+ channels but that the majority of cloned K+

channel a subunits display noninactivating currents uponexpression. They postulated that the "standard" configu-ration of A-type channels in vivo includes /3 subunits. It isintriguing for us to note that the only plant a subunitcDNA expressed in Xenopus oocytes displayed delayed-rectifier, noninactivating currents. In a recent review,Schroeder et al. (1994) alluded to the notion that K+ chan-nels in plants serve very different functions than in animalcells, i.e. in plants they should be designed for long-termK+ influx. They went on to postulate that it is sensible thatplant K+ channels lack an inactivation mechanism. How-ever, patch/clamp studies of native plant K+ channels doshow voltage-dependent inactivation (Schauf and Wilson,1987). Documentation of the presence of a K+ channel ftsubunit in plant cells as presented in this report raises thepossibility that more research may be required to resolvethe overall structure and inactivation properties of nativeplant K+ channel proteins.

Further work presented by Rettig et al. (1994) demon-strated that the extreme N-terminal portion of Kvj31 wascritical for a subunit inactivation; this section of Kv|31 isabsent from rat Kv|32, bovine Kv/32, and the KAB1 sequencepresented here. Co-expression of rat Kv/32 did not alterinactivation profiles of K+ channel a subunits (Rettig et al.,1994). Our KAB1 sequence is similar to bovine and ratKvj32. It is unclear, therefore, whether rat and bovine Kv/32

and KAB1 act in vivo to affect gating of K+ channel asubunits. However, even though the role KAB1 plays aspart of K+ channels in vivo remains unresolved, it is un-equivocally homologous to bovine Kvj32 (Fig. 1); bovineKv/32 has been thoroughly documented to be a structuralcomponent of native animal K+ channel proteins.

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 thalianapotassium channel in S. cerevisiae. Proc Natl Acad Sci USA 89:3736-3740

Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA miniprepa-ration: version II. Plant Mol Biol Rep 1: 19-21

Dolly JO, Rettig J, Scott VES, Parcej DN, Wittka R, Sewing S,Pongs O (1994) Oligomeric and subunit structures of neuronalvoltage-sensitive K+ channels. Structure and regulation of cat-ion channels. Biochem Soc Trans 22: 473^478

Jan LY, Jan YN (1994) Potassium channels and their evolvinggates. Nature 371: 119-122

Kyte J, Doolittle RF (1982) A simple method for displaying thehydropathic character of a protein. J Mol Biol 157: 105-132

Muniz ZM, Diniz CR, Dolly JO (1990) Characterisation of bindingsites for 8-dendrotoxin in guinea-pig synaptosomes: relationshipto acceptors for the K+ channel probe a-dendrotoxin. J Neuro-chem 54: 343-346

Muniz ZM, Parcej DN, Dolly JO (1992) Biochemistry 31: 12297-12303

Parcej DN, Dolly JO (1989) Dendrotoxin acceptor from bovinesynaptic plasma membranes. Binding properties, purificationand subunit composition of a putative constituent of certainvoltage-activated K+ channels. Biochem J 257: 899-903

Parcej DN, Scott VES, Dolly JO (1992) Oligomeric properties ofa-dendrotoxin-sensitive potassium ion channels purified frombovine brain. Biochemistry 31: 11084-11088

Rettig J, Helnemann SH, Wunder F, Lorra C, Parcej DN, DollyJO, Pongs O (1994) Inactivation properties of voltage-gated K +

channels altered by presence of /3-subunit. Nature 369: 289-294Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A

Laboratory Manual. Cold Spring Harbor Press, Cold SpringHarbor, NY, pp 9.31-9.58

Schachtman DP, Schroeder JI, Lucas WJ, Anderson JA, Gaber RF(1992) Expression of an inward-rectifying potassium channel bythe Arabidopsis KAT1 cDNA. Science 258: 1654-1658

Schauf CL, Wilson KJ (1987) Properties of single K+ and Cl~channels in Asclepias tuberosa protoplasts. Plant Physiol 85:413-418

Schroeder JI, Ward JM, Gassman W (1994) Perspectives on thephysiology and structure of inward-rectifying K+ channels inhigher plants: biophysical implications for K+ uptake. Annu RevBiophys Biomol Struct 23: 441-471

Scott VES, Parcej DN, Keen JN, Findlay JBC, Dolly JO (1990)a-Dendrotoxin acceptor from bovine brain is a K+ channel pro-tein. J Biol Chem 265: 20094-20097

Scott VES, Rettig J, Parcej DN, Keen JN, Findaly JBC, Pongs O(1994) Primary structure of a ft subunit of a-dendrotoxin-sensi-tive K+ channels from bovine brain. Proc Natl Acad Sci USA 91:1637-1641

Sentenac H, Bonneaud N, Minet M, Lacroute F, Salmon J-M,Gaynard F, Grignon C (1992) Cloning and expression in yeast ofa plant potassium ion transport system. Science 256: 683-685

Southern EM (1975) Detection of specific sequences among DNAfragments separated by gel electrophoresis. J Mol Biol 98:503-517

Sussman MR (1992) Shaking Arabidopsis thaliana. Science 256: 619

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evidence that plant k+ channel proteins have - plant physiology

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