Download Evidence That Plant K+ Channel Proteins Have

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
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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).
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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.
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^•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
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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.