The K+ Channel KZM1 Mediates Potassium Uptake into the Phloem and Guard Cells of the C4 Grass Zea mays

Artigo Acesso aberto Revisado por pares

The K+ Channel KZM1 Mediates Potassium Uptake into the Phloem and Guard Cells of the C4 Grass Zea mays

2003; Elsevier BV; Volume: 278; Issue: 19 Linguagem: Inglês

10.1074/jbc.m212720200

ISSN

1083-351X

Autores

Katrin Philippar, Kai Büchsenschütz, Maike Abshagen, Ines Fuchs, Dietmar Geiger, Benoı̂t Lacombe, Rainer Hedrich,

Tópico(s)

Aluminum toxicity and tolerance in plants and animals

Resumo

In search of K+ channel genes expressed in the leaf of the C4 plant Zea mays, we isolated the cDNA of KZM1 (forK+ channel Zeamays 1). KZM1 showed highest similarity to the Arabidopsis K+ channels KAT1 and KAT2, which are localized in guard cells and phloem. When expressed in Xenopus oocytes, KZM1 exhibited the characteristic features of an inward-rectifying, potassium-selective channel. In contrast to KAT1- and KAT2-type K+ channels, however, KZM1 currents were insensitive to external pH changes. Northern blot analyses identified the leaf, nodes, and silks as sites ofKZM1 expression. Following the separation of maize leaves into epidermal, mesophyll, and vascular fractions, quantitative real-time reverse transcriptase-PCR allowed us to localizeKZM1 transcripts predominantly in vascular strands and the epidermis. Cell tissue separation and KZM1 localization were followed with marker genes such as the bundle sheath-specific ribulose-1,5-bisphosphate carboxylase, the phloem K+channel ZMK2, and the putative sucrose transporterZmSUT1. When expressed in Xenopus oocytes, ZmSUT1 mediated proton-coupled sucrose symport. Coexpression of ZmSUT1 with the phloem K+ channels KZM1 and ZMK2 revealed that ZMK2 is able to stabilize the membrane potential during phloem loading/unloading processes and KZM1 to mediate K+ uptake. During leaf development, sink-source transitions, and diurnal changes,KZM1 is constitutively expressed, pointing to a housekeeping function of this channel in K+ homeostasis of the maize leaf. Therefore, the voltage-dependent K+-uptake channel KZM1 seems to mediate K+retrieval and K+ loading into the phloem as well as K+-dependent stomatal opening. In search of K+ channel genes expressed in the leaf of the C4 plant Zea mays, we isolated the cDNA of KZM1 (forK+ channel Zeamays 1). KZM1 showed highest similarity to the Arabidopsis K+ channels KAT1 and KAT2, which are localized in guard cells and phloem. When expressed in Xenopus oocytes, KZM1 exhibited the characteristic features of an inward-rectifying, potassium-selective channel. In contrast to KAT1- and KAT2-type K+ channels, however, KZM1 currents were insensitive to external pH changes. Northern blot analyses identified the leaf, nodes, and silks as sites ofKZM1 expression. Following the separation of maize leaves into epidermal, mesophyll, and vascular fractions, quantitative real-time reverse transcriptase-PCR allowed us to localizeKZM1 transcripts predominantly in vascular strands and the epidermis. Cell tissue separation and KZM1 localization were followed with marker genes such as the bundle sheath-specific ribulose-1,5-bisphosphate carboxylase, the phloem K+channel ZMK2, and the putative sucrose transporterZmSUT1. When expressed in Xenopus oocytes, ZmSUT1 mediated proton-coupled sucrose symport. Coexpression of ZmSUT1 with the phloem K+ channels KZM1 and ZMK2 revealed that ZMK2 is able to stabilize the membrane potential during phloem loading/unloading processes and KZM1 to mediate K+ uptake. During leaf development, sink-source transitions, and diurnal changes,KZM1 is constitutively expressed, pointing to a housekeeping function of this channel in K+ homeostasis of the maize leaf. Therefore, the voltage-dependent K+-uptake channel KZM1 seems to mediate K+retrieval and K+ loading into the phloem as well as K+-dependent stomatal opening. reverse transcriptase rapid amplification of cDNA ends 2-morpholinoethanesulfonic acid sodium acetate ribulose-1,5-bisphosphate carboxylase small subunit phosphoenolpyruvate carboxylase Since the first isolation of a plant K+ channel gene 10 years ago, plant science has focused on their cell-specific localization and structure-function relationship. Therefore, new insights into the physiological role of the different K+channel genes have been gained. The Arabidopsis thalianagenome contains at least 15 K+ channel genes (1Mäser P. Thomine S. Schroeder J.I. Ward J.M. Hirschi K. Sze H. Talke I.N. Amtmann A. Maathuis F.J.M. Sanders D. Harper J.F. Tchieu J. Gribskov M. Persans M.W. Salt D.E. Kim S.A. Guerinot M.L. Plant Physiol. 2001; 126: 1646-1667Crossref PubMed Scopus (969) Google Scholar). Among them, the Shaker family of ArabidopsisK+ channels consists of nine members (for review see Ref.2Very A.A. Sentenac H. Trends Pharmacol. Sci. 2002; 7: 168-175Abstract Full Text Full Text PDF Scopus (167) Google Scholar). According to their localization, structure, and function, these genes can be assigned to different subfamilies. In 1992, the first plant K+ channel genes isolated were AKT1 andKAT1 (3Anderson J.A. Huprikar S.S. Kochian L.V. Lucas W.J. Gaber R.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3736-3740Crossref PubMed Scopus (559) Google Scholar, 4Sentenac H. Bonneaud N. Minet M. Lacroute F. Salmon J.M. Gaymard F. Grignon C. Science. 1992; 256: 663-665Crossref PubMed Scopus (569) Google Scholar). Both proteins represent K+-uptake channels (5Gaymard F. Cerutti M. Horeau C. Lemaillet G. Urbach S. Ravallec M. Devauchelle G. Sentenac H. Thibaud J.B. J. Biol. Chem. 1996; 271: 22863-22870Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 6Schachtman D.P. Schroeder J.I. Lucas W.J. Anderson J.A. Gaber R.F. Science. 1994; 258: 1654-1658Crossref Scopus (365) Google Scholar). AKT1-like channels are involved in K+uptake into growing roots (AKT1) (7Dennison K.L. Robertson W.R. Lewis B.D. Hirsch R.E. Sussman M.R. Spalding E.P. Plant Physiol. 2001; 127: 1012-1019Crossref PubMed Scopus (100) Google Scholar, 8Hirsch R.E. Lewis B.D. Spalding E.P. Sussman M.R. Science. 1998; 280: 918-921Crossref PubMed Scopus (562) Google Scholar) and pollen tubes (SPIK) (9Mouline K. Very A.A. Gaymard F. Boucherez J. Pilot G. Devic M. Bouchez D. Thibaud J.B. Sentenac H. Genes Dev. 2002; 16: 339-350Crossref PubMed Scopus (191) Google Scholar), and KAT1 plays a role in Arabidopsis guard cells (10Kwak J.M. Murata Y. Baizabal-Aguirre V.M. Merrill J. Wang M. Kemper A. Hawke S.D. Tallman G. Schroeder J.I. Plant Physiol. 2001; 127: 473-485Crossref PubMed Scopus (159) Google Scholar, 11Nakamura R.L. McKendree W.L.J. Hirsch R.E. Sedbrook J.C. Gaber R.F. Sussman M.R. Plant Physiol. 1995; 109: 371-374Crossref PubMed Scopus (210) Google Scholar, 12Szyroki A. Ivashikina N. Dietrich P. Roelfsema M.R.G. Ache P. Reintanz B. Deeken R. Godde M. Felle H. Steinmeyer R. Palme K. Hedrich R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2917-2921Crossref PubMed Scopus (200) Google Scholar). Recently, the inward rectifier KAT2 could be characterized as the closest relative to KAT1 (13Pilot G. Lacombe B. Gaymard F. Cherel I. Boucherez J. Thibaud J.B. Sentenac H. J. Biol. Chem. 2001; 276: 3215-3221Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). KAT2 is expressed in guard cells, too, but in contrast to KAT1,KAT2 transcripts were identified in the phloem parenchyma of the leaf. A coding sequence of the channel gene AKT5 (AKT1 subfamily) could be isolated from hypocotyl tissue, 1S. Scheuermann, unpublished results. but its function still remains unknown. The AtKC1 gene splits into another subfamily and together with AKT1 subunits seems to generate the functional properties of the root hair K+-influx channel (14Ivashikina N. Becker D. Ache P. Meyerhoff O. Felle H.H. Hedrich R. FEBS Lett. 2001; 508: 463-469Crossref PubMed Scopus (154) Google Scholar, 15Reintanz B. Szyroki A. Ivashikina N. Ache P. Godde M. Becker D. Palme K. Hedrich R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4079-4084Crossref PubMed Scopus (186) Google Scholar). In contrast to the mentioned inward rectifiers within the AKT1 and KAT1 family, members of the AKT2/3 subfamily are characterized by weak voltage dependence, a Ca2+ and H+block, and seem to control phloem function (16Deeken R. Sanders C. Ache P. Hedrich R. Plant J. 2000; 23: 285-290Crossref PubMed Google Scholar, 17Deeken R. Geiger D. Fromm J. Koroleva O. Ache P. Langenfeld-Heyser R. Sauer N. Bennett M. Hedrich R. Planta. 2002; 216: 334-344Crossref PubMed Scopus (209) Google Scholar, 18Lacombe B. Pilot G. Michard E. Gaymard F. Sentenac H. Thibaud J.B. Plant Cell. 2000; 12: 837-851PubMed Google Scholar, 19Marten I. Hoth S. Deeken R. Ache P. Ketchum K.A. Hoshi T. Hedrich R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7581-7586Crossref PubMed Scopus (165) Google Scholar). The K+channel genes SKOR, localized in xylem vessels of the root (20Gaymard F. Pilot G. Lacombe B. Bouchez D. Bruneau D. Boucherez J. Michaux-Ferriere N. Thibaud J.B. Sentenac H. Cell. 1998; 94: 647-655Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar), and GORK, in guard cells, vasculature, and roots (14Ivashikina N. Becker D. Ache P. Meyerhoff O. Felle H.H. Hedrich R. FEBS Lett. 2001; 508: 463-469Crossref PubMed Scopus (154) Google Scholar, 21Ache P. Becker D. Ivashikina N. Dietrich P. Roelfsema M.R.G. Hedrich R. FEBS Lett. 2000; 486: 93-98Crossref PubMed Scopus (273) Google Scholar), build the subfamily of outward-rectifying K+ channels in Arabidopsis. Because the Arabidopsis genome reveals the complete set ofShaker-like K+ channel genes in plants, we can assign the orthologs from different plant species to the respective subfamilies. So far K+ channel genes have been isolated from 12 different plants (compare with Ref. 22Pratelli R. Lacombe B. Torregrosa L. Gaymard F. Romieu C. Thibaud J.B. Sentenac H. Plant Physiol. 2002; 128: 564-577Crossref PubMed Scopus (60) Google Scholar) including the C4 plant Zea mays. In maize, the two K+ channel genes ZMK1 and ZMK2, isolated from the coleoptile, belong to the AKT1- and AKT2/3-type subfamilies, respectively (23Philippar K. Fuchs I. Lüthen H. Hoth S. Bauer C. Haga K. Thiel G. Ljung K. Sandberg G. Böttger M. Becker D. Hedrich R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12186-12191Crossref PubMed Scopus (247) Google Scholar). ZMK1 is involved in auxin-induced K+ uptake, coleoptile growth, and tropisms. ZMK2 displays the voltage-independent features of the AKT2/3-type K+ channels and thus seems to serve phloem-associated functions (24Bauer C.S. Hoth S. Haga K. Philippar K. Aoki N. Hedrich R. Plant J. 2000; 24: 139-145Crossref PubMed Scopus (56) Google Scholar). Besides rice, the maize plant is not only a model system for monocotyledonous crops but C4 photosynthesis as well. This involves a special anatomic feature called Kranz anatomy: mesophyll cells, involved in the pre-fixation of CO2, transport C4 compounds to the bundle sheath cells, which surround the vascular strands and finally fix CO2 in the Calvin cycle (reviewed in Ref. 25Nelson T. Langdale J.A. Annu. Rev. Plant Physiol. Plant. Mol. Biol. 1992; 43: 25-47Crossref Scopus (127) Google Scholar). Due to this cell and chloroplast dimorphism, C4 plants are characterized by a better water-use efficiency than C3 plants. The carbohydrate transport between mesophyll, bundle sheath, and vascular parenchyma cells of the maize leaf is accomplished by numerous plasmodesmata (26Botha C.E.J. Cross R.H.M. Van Bel A.J.E. Peter C.I. Protoplasma. 2000; 214: 65-72Crossref Scopus (75) Google Scholar, 27Russin W.A. Evert R.F. Vanderveer P.J. Sharkey T.D. Briggs S.P. Plant Cell. 1996; 8: 645-658Crossref PubMed Google Scholar, 28Stitt M. Plant Cell. 1996; 8: 565-571Crossref Google Scholar). The loading of sucrose from the vascular parenchyma to the thin-walled sieve tubes, representing the site of assimilate export, however, is thought to involve an apoplastic step (reviewed in Refs. 29Prioul J.L. Zamski E. Schaffer A.A. Photoassimilate Distribution in Plants and Crops, Source-Sink Relationships. Marcel Dekker, Inc., New York1996: 549-594Google Scholarand 30Van Bel A.J.E. Annu. Rev. Plant Physiol. Plant. Mol. Biol. 1993; 44: 253-281Crossref Scopus (204) Google Scholar). To characterize the sugar import machinery, Aoki et al. (31Aoki N. Hirose T. Takahashi S. Ono K. Ishimaru K. Ohsugi R. Plant Cell Physiol. 1999; 40: 1072-1078Crossref PubMed Scopus (98) Google Scholar) isolated ZmSUT1, a putative sucrose transporter from source leaves of maize. By heterologous expression inXenopus oocytes, we showed that ZmSUT1 indeed represents a sucrose/H+ symporter under the voltage control of the AKT2/3 ortholog ZMK2. Because the AKT2/3-type channels such as AKT2/3 fromArabidopsis, VFK1 from Vicia faba, and ZMK2 fromZ. mays seem to play an important role in the control of phloem sucrose loading and unloading (16Deeken R. Sanders C. Ache P. Hedrich R. Plant J. 2000; 23: 285-290Crossref PubMed Google Scholar, 17Deeken R. Geiger D. Fromm J. Koroleva O. Ache P. Langenfeld-Heyser R. Sauer N. Bennett M. Hedrich R. Planta. 2002; 216: 334-344Crossref PubMed Scopus (209) Google Scholar, 24Bauer C.S. Hoth S. Haga K. Philippar K. Aoki N. Hedrich R. Plant J. 2000; 24: 139-145Crossref PubMed Scopus (56) Google Scholar, 32Ache P. Becker D. Deeken R. Dreyer I. Weber H. Fromm J. Hedrich R. Plant J. 2001; 27: 571-580Crossref PubMed Google Scholar), we here studied K+ channels expressed in the dimorphic structure of the maize leaf. In addition to ZMK2 (23Philippar K. Fuchs I. Lüthen H. Hoth S. Bauer C. Haga K. Thiel G. Ljung K. Sandberg G. Böttger M. Becker D. Hedrich R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12186-12191Crossref PubMed Scopus (247) Google Scholar), we isolated the cDNA ofKZM1. KZM1 represents the maize ortholog toKAT2 from Arabidopsis. LikeKAT2 we found this new maize K+ channel gene expressed in vascular/bundle sheath strands as well as guard cell- and subsidiary cell-enriched epidermal fractions of the maize leaf. However, KZM1 is characterized by unique functional properties that enabled us to discriminate between the function of KAT2 in the dicotyledonous plant Arabidopsis and KZM1 in the monocotyledonous C4 plant maize. The K+-uptake channel KZM1 is able to mediate phloem K+ loading and retrieval as well as K+-dependent stomatal movement. The function of the inward-rectifier KZM1 in combination with ZMK2 and the sucrose/H+ symporter ZmSUT1 as well as its expression pattern point to a housekeeping function of KZM1 for K+homeostasis in the phloem of the maize leaf. Cloning, Northern blot procedures, and quantitative real-time RT2-PCR analysis were performed on tissues isolated from maize plants (Z. mays L., hybrid corn cv. “Oural FA0230,” Deutsche Saatveredelung, Lippstadt, Germany). Seeds were sown in soil and grown in a greenhouse with a 16-h light (25 °C) and 8-h dark (18 °C) cycle. The white light used had a photon-flux density of 210 ॖmol·m−2·s−1 (LiCOR Quantum Sensor LI-250, Walz GmbH, Effeltrich, Germany). After harvesting, all maize tissues were stored in liquid nitrogen prior to RNA extraction. The age of plants or organs is denoted in days after sowing. Degenerated oligonucleotide primers, directed toward homologous regions of known plant inward-rectifying K+ channels, were used to amplify a corresponding region of potassium channels from reverse-transcribed maize leaf RNA (RT-PCR). By using the SMART RACE cDNA Amplification kit (Clontech, Heidelberg, Germany) in combination with gene-specific primers, we amplified overlapping N- and C-terminal K+ channel fragments according to the RACE technique. The corresponding full-length cDNA was generated in a single PCR step using primers flanking the 5′- and 3′-ends of the coding sequence of KZM1 and ligated into pCRII-TOPO TA vector (Invitrogen). Besides the N- and C-terminal clones ofKZM1, three identical full-length clones of the channel cDNA were sequenced using the LiCOR 4200 sequencer (LiCOR, Bad Homburg, Germany). Total RNA was isolated from the respective maize organs using the Plant RNeasy Extraction kit (Qiagen, Hilden, Germany). Poly(A)+ RNA was purified from total RNA using Dynabeads (Dynal, Hamburg, Germany) and subjected to Northern blot analysis as described (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). The blotted poly(A)+ RNA was hybridized against 32P-radiolabeled full-length cDNA probes of the K+ channel genes KZM1 andZMK2 as described in Philippar et al. (23Philippar K. Fuchs I. Lüthen H. Hoth S. Bauer C. Haga K. Thiel G. Ljung K. Sandberg G. Böttger M. Becker D. Hedrich R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12186-12191Crossref PubMed Scopus (247) Google Scholar). For the sucrose transporter ZmSUT1, a 339-bp-long 3′-terminal cDNA fragment, amplified between the primers ZmSUT LCfw (5′-cccacaaaggcaaac-3′) and ZmSUT LCrev (5′-tggtgtgggtgacg-3′), served as a probe. Each probe exhibited specific signals at 2.5 kb forKZM1, 2.8 kb for ZMK2, and 2.0 kb forZmSUT1. To standardize transcript abundance, 15 ng of dotted poly(A)+ was hybridized against a [γ-32P]dATP end-labeled oligo(dT) probe as described (23Philippar K. Fuchs I. Lüthen H. Hoth S. Bauer C. Haga K. Thiel G. Ljung K. Sandberg G. Böttger M. Becker D. Hedrich R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12186-12191Crossref PubMed Scopus (247) Google Scholar). Tissue from the last fully developed leaf of 5-week-old maize plants was separated into epidermis, mesophyll cells, and vascular strands by a procedure modified according to Keunecke and Hansen (34Keunecke M. Hansen U.P. Planta. 2000; 210: 792-800Crossref PubMed Scopus (14) Google Scholar). The central vascular strand was excised, and the lower epidermis was collected in 1 mm CaCl2, 5 mm Mes/KOH, pH 6.5, adjusted with mannitol to 530 mosmol kg−1, and frozen in liquid nitrogen for mRNA extraction. To test the vitality of epidermal cells before freezing, an aliquot of the epidermal fraction was stained with neutral red (see Fig. 4). To isolate mesophyll protoplasts, the remaining leaf sections were incubated for 90 min at 30 °C in enzyme solution containing 1.57 cellulase (Cellulase R-10, Yakult Honsha Co., Tokyo, Japan), 27 pektinase (Sigma), 10 mm KCl, 10 mm Mes/KOH, pH 6.2, adjusted withd-sorbitol to 480 mosmol kg−1. The digestion was stopped before the bundle sheath cells were released from the vascular strands. Isolated vascular/bundle sheath strands were pooled and frozen in liquid nitrogen. Mesophyll protoplasts were sedimented at 60 × g for 5 min at 4 °C and frozen in liquid nitrogen. For real-time RT-PCR experiments, total RNA from the fractionated maize leaves was isolated using the Plant RNeasy Extraction kit (Qiagen, Hilden, Germany). To minimize DNA contaminations, mRNA was purified twice with the Dynabeads mRNA Direct kit (Dynal, Hamburg, Germany), or total RNA was subjected to digestion with RNase-free DNase and purified by phenol/chloroform extraction (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Mesophyll protoplast mRNA was directly purified with the Dynabeads mRNA Direct kit (Dynal). First-strand cDNA synthesis and quantitative real-time RT-PCR were performed as described before (12Szyroki A. Ivashikina N. Dietrich P. Roelfsema M.R.G. Ache P. Reintanz B. Deeken R. Godde M. Felle H. Steinmeyer R. Palme K. Hedrich R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2917-2921Crossref PubMed Scopus (200) Google Scholar) using a LightCycler (Roche Molecular Biochemicals). The following K+ channel-specific primers were used: KZM1 LCfw (5′-aagaagcatggttgttac-3′), KZM1 LCrev (5′-tgaaaccaaagaagtctc-3′), ZMK2 LCfw (5′-gacggctcaggttcag-3′), and ZMK2 LCrev (5′-gagaaggcgttgatcg-3′). For detection of the coding sequence of the small subunit of the ribulose-1,5-bisphosphate carboxylase (ZmRuBPCsu, GenBankTM accession number X06535) and the 3′-untranslated region of the C4 form phosphoenolpyruvate carboxylase (ZmC4-PEPC, GenBankTM accession number X15238), we used the primer pairs RuBPCssu LCfw (5′-caacaagaagttcgagacg-3′), RuBPCssu LCrev (5′-cgggtaggatttgatggc-3′), and C4-PEPC LCfw (5′-ggcttctcttcactcacc-3′), C4-PEPC LCrev (5′-tccaatgggctgggata-3′), respectively. All quantifications were normalized to the signal of actin cDNA fragments generated by the primers ZmAct 81/83fw (5′-acacagtgccaatct-3′) and ZmAct 81/83rev (5′-actgagcacaatgttac-3′), which amplified cDNA from the maize actins ZmAct 81 (GenBankTM accession number AAB40106) and ZmAct 83 (GenBankTM accession number AAB40105). The relative amount of channel cDNA was calculated from the correlation 2(nactin −nchannel) with n = threshold cycle of the respective PCR product. To identify contaminating genomic DNA, the primers for ZMK2 were selected to flank an intron. For heterologous expression in Xenopus laevisoocytes, the cDNAs of KZM1 in pCRII andZmSUT1 in pBS SK(−) were subcloned asXhoI/SpeI and BamHI/XhoI fragments, respectively, into the pGEMHE vector (35Liman E.R. Tytgat J. Hess P. Neuron. 1992; 9: 861-871Abstract Full Text PDF PubMed Scopus (983) Google Scholar). Expression ofZMK2 in oocytes was performed as described (23Philippar K. Fuchs I. Lüthen H. Hoth S. Bauer C. Haga K. Thiel G. Ljung K. Sandberg G. Böttger M. Becker D. Hedrich R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12186-12191Crossref PubMed Scopus (247) Google Scholar). The respective cRNA was generated by in vitro transcription (T7-Megascript kit, Ambion Inc., Austin, TX) and injected intoXenopus oocytes (CRBM, CNRS, Montpellier, France) using a PicospritzerII microinjector (General Valve, Fairfield, NJ). Two to 6 days following injection, double-electrode voltage clamp recordings were performed with a Turbotec-01C amplifier (NPI Instruments, Tamm, Germany). The electrodes were filled with 3 m KCl and had typical input resistance of about 2–4 megaohm. Solutions were composed of 30 mm KCl, 2 mm MgCl2, 1 mm CaCl2, and 10 mmTris/Mes, pH 7.5, 10 mm Mes/Tris, pH 5.6, and 10 mm citrate/Tris, pH 4.5, respectively. Acidification of the cytosolic pH was accomplished by perfusion with 30 mm KCl, 2 mm MgCl2, 1 mm CaCl2and 10 mm Mes/Tris, pH 5.6, as well as 10 mmNaAc. The control solution contained 10 mm NaCl instead of NaAc. When recording KZM1-mediated currents at 100, 30, 10, and 3 mm external K+ concentrations, the ionic strength was adjusted with Na+. In K+-free solutions, K+ was substituted with Na+ or Li+ as indicated. All media were adjusted to a final osmolality of 215–235 mosmol kg−1 withd-sorbitol. Analyses of voltage and pH dependence were performed as described previously (19Marten I. Hoth S. Deeken R. Ache P. Ketchum K.A. Hoshi T. Hedrich R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7581-7586Crossref PubMed Scopus (165) Google Scholar, 36Hoth S. Dreyer I. Dietrich P. Becker D. Müller-Röber B. Hedrich R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4806-4810Crossref PubMed Scopus (113) Google Scholar). Membrane potential measurements with ZmSUT1-expressing oocytes and coexpression of ZmSUT1 with KZM1 and ZMK2 were performed as described for AtSUC2, KAT2, and AKT2/3 in Deeken et al. (17Deeken R. Geiger D. Fromm J. Koroleva O. Ache P. Langenfeld-Heyser R. Sauer N. Bennett M. Hedrich R. Planta. 2002; 216: 334-344Crossref PubMed Scopus (209) Google Scholar). For patch clamp experiments, devitellinized oocytes were placed in a bath solution containing 100 mm KCl, 2 mm MgCl2, 1 mm CaCl2, and 10 mm Tris/Mes, pH 7.5. Pipettes were filled with solution containing 100 mmKCl, 2 mm MgCl2, 1 mmCaCl2, and 10 mm Tris/Mes, pH 7.5. Currents were recorded in the cell-attached configuration using an EPC-9 amplifier (HEKA, Lambrecht, Germany) as described previously (37Hedrich R. Moran O. Conti F. Busch H. Becker D. Gambale F. Dreyer I. Kuech A. Neuwinger K. Palme K. Eur. Biophys. J. 1995; 24: 107-115Crossref PubMed Scopus (84) Google Scholar). To study the role of K+ channels in C4 leaves, characterized by Kranz anatomy, we isolatedKZM1 from maize leaf cDNA via RT-PCR and RACE techniques. The cloning strategy took advantage of highly conserved regions in the Shaker gene family of plant K+channels (for review see Ref. 2Very A.A. Sentenac H. Trends Pharmacol. Sci. 2002; 7: 168-175Abstract Full Text Full Text PDF Scopus (167) Google Scholar). Sequence analysis of the open reading frame of the KZM1 cDNA (2274 bp) revealed the basic features of the KAT1 subfamily (Fig.1A) as follows: six putative transmembrane domains (S1–S6) with a proposed voltage sensor in segment 4 and a K+-selective pore (P), formed by the amphiphilic linker between S5 and S6 (for structure-function analysis of plant K+ channels see Refs. 38Hoth S. Geiger D. Becker D. Hedrich R. Plant Cell. 2001; 13: 943-952PubMed Google Scholar and 39Geiger D. Becker D. Lacombe B. Hedrich R. Plant Cell. 2002; 14: 1859-1868Crossref PubMed Scopus (33) Google Scholar). The deduced KZM1 protein spans 758 amino acids with a predicted molecular mass of 86.7 kDa. When compared on the amino acid level to theArabidopsis K+ channels of the Shakerfamily, KZM1 showed highest similarity to KAT2 (487 identity, Ref. 13Pilot G. Lacombe B. Gaymard F. Cherel I. Boucherez J. Thibaud J.B. Sentenac H. J. Biol. Chem. 2001; 276: 3215-3221Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar) and KAT1 (477 identity, Ref. 3Anderson J.A. Huprikar S.S. Kochian L.V. Lucas W.J. Gaber R.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3736-3740Crossref PubMed Scopus (559) Google Scholar), whereas the identity to the previously identified maize K+ channels ZMK1 and ZMK2 (23Philippar K. Fuchs I. Lüthen H. Hoth S. Bauer C. Haga K. Thiel G. Ljung K. Sandberg G. Böttger M. Becker D. Hedrich R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12186-12191Crossref PubMed Scopus (247) Google Scholar) was only 36 and 347, respectively. Thus, KZM1 represents a member of the KAT1 subfamily of plant K+ channels (Fig.1B). The proposed cytoplasmic C terminus of KZM1 contains a region, which shares structural homologies to cyclic nucleotide binding domains (Fig. 1A). In contrast to ZMK2 (five ankyrin repeats), the sequence of KZM1 did not contain an ankyrin binding domain (Fig. 1A), a feature that is conserved among K+ channels of the KAT1 subfamily (1Mäser P. Thomine S. Schroeder J.I. Ward J.M. Hirschi K. Sze H. Talke I.N. Amtmann A. Maathuis F.J.M. Sanders D. Harper J.F. Tchieu J. Gribskov M. Persans M.W. Salt D.E. Kim S.A. Guerinot M.L. Plant Physiol. 2001; 126: 1646-1667Crossref PubMed Scopus (969) Google Scholar), beside the SIRK protein from Vitis vinifera (22Pratelli R. Lacombe B. Torregrosa L. Gaymard F. Romieu C. Thibaud J.B. Sentenac H. Plant Physiol. 2002; 128: 564-577Crossref PubMed Scopus (60) Google Scholar) and KPT1 from Populus tremula (GenBankTMaccession number AJ344623, for details see “Discussion”). In the 5′-region of the open reading frame of KZM1, we could identify two possible translational start positions (“ATG”), a structural element also found with the Arabidopsis orthologKAT2 (Fig. 1A). In addition, plant-specific hydrophobic and acidic C-terminal domains, involved in plant K+ channel clustering (40Ehrhardt T. Zimmermann S. Müller-Röber B. FEBS Lett. 1997; 409: 166-170Crossref PubMed Scopus (70) Google Scholar, 45Daram P. Urbach S. Gaymard F. Sentenac H. Cherel I. EMBO J. 1997; 16: 3455-3463Crossref PubMed Scopus (117) Google Scholar), could be identified. Based on Southern blot analysis with maize DNA, we characterizedKZM1 as a single copy gene within the maize genome (not shown). Besides KZM1 we could also identifyKZM2, 3K. Büchsenschütz, unpublished results. the second maize member of the KAT1 subfamily (Fig. 1B), most likely representing the ortholog to KAT1 fromArabidopsis. When expressed in Xenopus oocytes, the gene product of KZM1 showed the characteristic properties of a voltage-dependent, inward-rectifying plant K+channel (Fig. 2). In two-electrode voltage clamp experiments, KZM1 activated upon hyperpolarization to membrane potentials negative to −60 mV (Fig. 2A). The steady-state current-voltage curve of the data shown in Fig.2A underlines the strong inward rectification of KZM1 (Fig.2B). From activation curve analyses, a half-maximal activation voltage U12 of −105.4 ± 6.9 mV (n = 5) was calculated. Recordings in the cell-attached patch clamp configuration allowed us to resolve single KZM1 channel-fluctuations (Fig. 2C). The channel amplitude and time-dependent activity increased with increasing negative voltages. From the current-voltage relationship of the single channels (Fig. 2D), a unitary conductance of 20 ± 0.7 pS (n = 3, mean ± S.E. with 100 mmK+ in the pipette) was deduced. Thus, KZM1 exhibits a 2–4-fold higher conductance than the previously characterized K+ channels of the KAT1 subfamily (6.7 pS for KAT2 (13Pilot G. Lacombe B. Gaymard F. Cherel I. Boucherez J. Thibaud J.B. Sentenac H. J. Biol. Chem. 2001; 276: 3215-3221Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar), 5 pS for KAT1 (37Hedrich R. Moran O. Conti F. Busch H. Becker D. Gambale F. Dreyer I. Kuech A. Neuwinger K. Palme K. Eur. Biophys. J. 1995; 24: 107-115Crossref PubMed Scopus (84) Google Scholar), 7 pS for KST1 (46Müller-Röber B. Ellenberg J. Provart N. Willmitzer L. Busch H. Becker D. Dietrich P. Hoth S. Hedrich R. EMBO J. 1995; 14: 2409-2416Crossref PubMed Scopus (187) Google Scholar), and 13 pS for SIRK (22Pratelli R. Lacombe B. Torregrosa L. Gaymard F. Romieu C. Thibaud J.B. Sentenac H. Plant Physiol. 2002; 128: 564-577Crossref PubMed Scopus (60) Google Scholar)). In agreement with a K+-selective channel, K+currents through KZM1 increased as a function of the external K+ concentration (not shown) with the current reversal potential following the Nernst potential for potassium (60.8 ± 4.7 mV per 10-fold change in external K+ concentration, Fig. 3A). Replacing K+ by Rb+ (100 mm) caused a drop in the inward current (at −150 mV, IRb+/IK+ = 0.190 ± 0.041,n = 4). Comparison of the reversal potential in either K+ or Rb+ solutions allowed us to determine the permeability ratioPRb/PK = 0.437 ± 0.085, n = 4. In contrast to other KAT1-like channels, the permeability ratios for Na+ and Li+ ions could not be determined, because KZM1 did not even conduct outward currents in N

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The K+ Channel KZM1 Mediates Potassium Uptake into the Phloem and Guard Cells of the C4 Grass Zea mays