Mori IC et al. (2018), A Cyclic Nucleotide-Gated Channel, HvCNGC2-3, I...

XB-ART-55144

Plants (Basel) 2018 Jul 26;73:. doi: 10.3390/plants7030061.

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A Cyclic Nucleotide-Gated Channel, HvCNGC2-3, Is Activated by the Co-Presence of Na⁺ and K⁺ and Permeable to Na⁺ and K⁺ Non-Selectively.

Mori IC , Nobukiyo Y , Nakahara Y , Shibasaka M , Furuichi T , Katsuhara M .

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Cyclic nucleotide-gated channels (CNGCs) have been postulated to contribute significantly in plant development and stress resistance. However, their electrophysiological properties remain poorly understood. Here, we characterized barley CNGC2-3 (HvCNGC2-3) by the two-electrode voltage-clamp technique in the Xenopus laevis oocyte heterologous expression system. Current was not observed in X. laevis oocytes injected with HvCNGC2-3 complementary RNA (cRNA) in a bathing solution containing either Na⁺ or K⁺ solely, even in the presence of 8-bromoadenosine 3',5'-cyclic monophosphate (8Br-cAMP) or 8-bromoguanosine 3',5'-cyclic monophosphate (8Br-cGMP). A weakly voltage-dependent slow hyperpolarization-activated ion current was observed in the co-presence of Na⁺ and K⁺ in the bathing solution and in the presence of 10 µM 8Br-cAMP, but not 8Br-cGMP. Permeability ratios of HvCNGC2-3 to K⁺, Na⁺ and Cl- were determined as 1:0.63:0.03 according to reversal-potential analyses. Amino-acid replacement of the unique ion-selective motif of HvCNGC2-3, AQGL, with the canonical motif, GQGL, resulted in the abolition of the current. This study reports a unique two-ion-dependent activation characteristic of the barley CNGC, HvCNGC2-3.

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	Figure 1. Phylogenetic tree of CNGC genes in plants. Barley CNGC genes are highlighted by boxes. Accession numbers of barley genes are described in Table S1. Arabidopsis Genome Initiative codes are available in Table S2. Rice Annotation Project identifiers are shown for rice CNGCs.
	Figure 2. Expression analysis of HvCNGC2-3 mRNA by reverse transcription-polymerase chain reaction (RT-PCR). DNA fragments of HvEF1α (A) and HvCNGC2-3 (C) amplified with RT-PCR from three independently prepared cDNA samples were analyzed by agarose gel electrophoresis. G, HvCNGC2-3 band amplified from genomic DNA of barley cultivar Haruna-Nijyo for comparison. Arrowhead indicates the position of HvCNGC2-3. M, DNA size marker ‘Without RT’ indicates PCR using the template without reverse-transcription reaction controls serving as a control experiment proving free of genomic DNA contamination in the samples using HvEF1α primers (B) and HvCNGC2-3 primers (D).
	Figure 3. Current/voltage relationship of HvCNGC2-3-expressing oocytes in Na+ and K+ media. Currents were recorded by the two-electrode voltage-clamp technique. HvCNGC2-3 cRNA-injected oocytes (open circles), water-injected control oocytes (closed circles). (A,B) bath solution containing a Na+ medium (96 mM NaCl); (C,D) bath solution containing a K+ medium (96 mM KCl). Oocytes were treated with (B,D) or without (A,C) 8Br-cAMP (10 μM) 30 min before measurement. Current values are means (n = 14 [panel A], 7 [panel B], 13 [panel C] and 7 [panel D] for water-injected control, and n = 6 [A], 6 [B], 13 [C] and 7 [D] for HvCNGC2-3) ± SD. I, membrane current. Vm, membrane potential. Asterisks indicate significant difference of the means between HvCNGC2-3 cRNA-injected oocytes and water-injected control oocytes (Student’s t-test, p < 0.05).
	Figure 4. Current/voltage relationship of HvCNGC2-3-expressing oocytes in a 1:1 mixture of Na+ and K+ media. Currents were recorded by the two-electrode voltage-clamp technique in a bathing solution containing 48 mM NaCl and 48 mM KCl in the absence of a pre-incubation with cyclic nucleoside monophosphates (cNMP-free, circles), and in the presence of a pre-incubation with 10 µM 8Br-cGMP (triangles) or 8Br-cAMP (squares) for 30 min. (A) representative raw recording traces; (B) current/voltage curve. Error bars indicate standard deviation (n = 7, 13 and 9 for cNMP-free, 8Br-cGMP and 8Br-cAMP, respectively). I, membrane current. Vm, membrane potential; (C) Po, apparent open probability fitted with modified Boltzmann equation.
	Figure 5. Current/voltage relationship of HvCNGC2-3-expressing oocytes in a 1:4 mixture of Na+ and K+ media. Currents were recorded by the two-electrode voltage-clamp technique in a bathing solution containing 19.2 mM NaCl and 76.8 mM KCl in the absence of a pre-incubation with cyclic nucleoside monophosphates (cNMP-free, circles), and in the presence of a pre-incubation with 10 µM 8Br-cGMP (triangles) or 8Br-cAMP (squares) for 30 min. (A) representative raw recording traces; (B) current/voltage curve. Error bars indicate standard deviation (n = 9, 13 and 6 for cNMP-free, cGMP and cAMP, respectively). I, membrane current. Vm, membrane potential.
	Figure 6. Current/voltage relationship of HvCNGC2-3-expressing oocytes in a gluconate-containing medium and N-methyl-d-glucamine-containing medium. Currents were analyzed with the two-electrode voltage-clamp technique. Oocytes were treated with 8Br-cAMP (10 μM) for 30 min before measurement. Oocytes expressing HvCNGC2-3 (open symbols) or water-injected controls (closed symbols) were bathed in a solution containing 48 mM KCl and 48 mM Na-gluconate (A) and 9.6 mM KCl, 9.6 mM NaCl and 76.8 mM N-methyl-d-glucamine-Cl [NMCG-Cl] (B). Current values are means (n = 9 for water-injected control, n = 19 for HvCNGC2-3 of gluconate-containing medium and n = 4 for HvCNGC2-3 of N-methyl-d-glucamine-containing medium) ± SD. Erev, reversal potential.
	Figure 7. Alignment of deduced amino acid sequences of the putative ion-selective pore-forming motif (G/AQGL) of group-II CNGSs and voltage-clamp analysis of oocytes expressing HvCNGC2-3A394G. (A) the ion-selective motif is highlighted by box. Numbers indicate the amino acid positions. Alanine residue in the ion-selective motif of HvCNGC2-3 and Phenylalanine residue in the ion-selective motif of HvCNGC2-2 are underlined. a Gene names of rice are according to Rice Annotation Project identifiers (URL: https://rapdb.dna.affrc.go.jp). Arabidopsis Genome Initiative codes of Arabidopsis CNGCs are shown in Table S2. Gene names of barley CNGCs were allocated by this study (for accession number of each CNGC gene of barley, see Table S1); (B) representative raw current traces. Bathing solution contained 48 mM KCl and 48 mM NaCl; (C) current–voltage relationship. Oocytes were treated with cNMP-free bathing solution (closed symbols) or with 8Br-cAMP bathing solution (10 μM, open symbols) 30 min before measurements. Current values are means (n = 12 for cNMP-free and n = 10 for 8Br-cAMP) ± SD. Asterisks indicate a significant difference of the means between oocytes treated with and without 8Br-cAMP (Student’s t-test, p < 0.05).

References [+] :

Abdel-Hamid, A suppressor screen of the chimeric AtCNGC11/12 reveals residues important for intersubunit interactions of cyclic nucleotide-gated ion channels. 2013, Pubmed