Tran STH et al. (2020), A Survey of Barley PIP Aquaporin Ionic Conducta...

XB-ART-57402

Int J Mol Sci 2020 Sep 27;2119:. doi: 10.3390/ijms21197135.

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A Survey of Barley PIP Aquaporin Ionic Conductance Reveals Ca2+-Sensitive HvPIP2;8 Na+ and K+ Conductance.

Tran STH , Horie T , Imran S , Qiu J , McGaughey S , Byrt CS , Tyerman SD , Katsuhara M .

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Some plasma membrane intrinsic protein (PIP) aquaporins can facilitate ion transport. Here we report that one of the 12 barley PIPs (PIP1 and PIP2) tested, HvPIP2;8, facilitated cation transport when expressed in Xenopus laevis oocytes. HvPIP2;8-associated ion currents were detected with Na+ and K+, but not Cs+, Rb+, or Li+, and was inhibited by Ba2+, Ca2+, and Cd2+ and to a lesser extent Mg2+, which also interacted with Ca2+. Currents were reduced in the presence of K+, Cs+, Rb+, or Li+ relative to Na+ alone. Five HvPIP1 isoforms co-expressed with HvPIP2;8 inhibited the ion conductance relative to HvPIP2;8 alone but HvPIP1;3 and HvPIP1;4 with HvPIP2;8 maintained the ion conductance at a lower level. HvPIP2;8 water permeability was similar to that of a C-terminal phosphorylation mimic mutant HvPIP2;8 S285D, but HvPIP2;8 S285D showed a negative linear correlation between water permeability and ion conductance that was modified by a kinase inhibitor treatment. HvPIP2;8 transcript abundance increased in barley shoot tissues following salt treatments in a salt-tolerant cultivar Haruna-Nijo, but not in salt-sensitive I743. There is potential for HvPIP2;8 to be involved in barley salt-stress responses, and HvPIP2;8 could facilitate both water and Na+/K+ transport activity, depending on the phosphorylation status.

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Genes referenced: arhgef40

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	Figure 1. Electrophysiological survey to test for HvPIP2 ion transport (A,B) Currentâ€“voltage relationships of X. leavis oocytes expressing each HvPIP2 in the presence of 86.4 mM NaCl and 9.6 mM KCl with 30 ÂµM Ca2+ (A) or 1.8 mM Ca2+ (B). A total of 10 ng of each HvPIP2 cRNA or water (control) was injected into X. laevis oocytes. (C) Relationships between the external free Ca2+ concentration and HvPIP2;8-mediated Na+ conductance in the presence of 86.4 mM NaCl and 9.6 mM KCl (R2 = 0.93). The free Ca2+ concentrations are given in Methods. A step pulse protocol of âˆ’120 mV to +30 mV with a 15 mV increment was applied on every oocyte. Ionic conductance was calculated based on the data obtained from V = âˆ’75 mV to âˆ’120 mV of the membrane potential. Data are the means Â± SE (n = 5 for A,C, and n = 7 for B).
	Figure 2. Monovalent alkaline cation selectivity of HvPIP2;8 and the effect of the interaction of K+ and Na+ on HvPIP2;8-mediated ion conductance activity. Currentâvoltage relationships obtained from oocytes either expressing HvPIP2;8 (A) or injected with water (B) HvPIP2;8 displays a different monovalent alkaline cation selectivity. Oocytes were successively immersed in bath solutions with a high calcium condition, supplemented with Na+, K+, Cs+, Rb+, and Li+ (as chloride salts) at the concentration of 96 mM. (C) Inhibition of HvPIP2;8-mediated Na+ transport by monovalent alkaline cations in the presence of 48 mM NaCl with 48 mM of each alkaline cation. (D) The effect of external Na+/K+ concentration ratios on the conductance of HvPIP2;8-expressing oocytes from V (membrane potential) = â75 mV to â120 mV. The total concentration of (Na + K) was constantly 96 mM. X. laevis oocytes were injected with 10 ng of HvPIP2;8 cRNA for the recording of the conductance in every experiment. Data are the means Â± SE (n = 7 to 8 for A, n = 5 for B, n = 4 to 5 for C, and n = 5 to 6 for D).
	Figure 3. HvPIP2;8-mediated Na+ transport is Clâ independent. (A) Currentâvoltage relationships obtained from oocytes either expressing HvPIP2;8 or injected with water in the presence of either 96 mM NaCl or 96 mM Na-gluconate. Inset: Expanded current voltage curves around the reversal potential. (B) Currentâvoltage relationships obtained from oocytes either expressing HvPIP2;8 or injected with water in the presence of either 96 mM NaCl or 96 mM Choline-Cl. All solutions contained 30 ÂµM Ca2+. X. laevis oocytes were injected with 10 ng of HvPIP2;8 cRNA. Data are the means Â± SE (n = 7 to 8).
	Figure 4. Effects of divalent cations on the ion current responses in oocytes expressing HvPIP2;8. (A) Effect of divalent cations on the ion currents of the HvPIP2;8-transporter; bath solutions with a high 1.8 mM Ca2+ background calcium conditions were successively replaced with either 1.8 mM Ca2+, Ba2+, Cd2+, and Mg2+ (as chloride salts), at concentrations of 86.4 mM NaCl and 9.6 mM KCl. (B) Box plot summary of the ionic conductance presented in (A); the ionic conductances were calculated from V = âˆ’75 mV to âˆ’120 mV. (C) Relief of Ca2+ inhibition by the addition of Mg2+ on the ion current responses in oocytes expressing HvPIP2;8; note the different range of the Y-axis from the plot in (A). (D) Box plot summary of the ionic conductance presented in (C). Steady-state currentâ€“voltage curves of the X. laevis oocytes injected with 10 ng of cRNA per oocyte were recorded. Currents from the oocytes injected with water were the negative controls from the same batch. Significant differences (p < 0.05) are indicated by different letters using one-way ANOVA with Duncanâ€™s multiple comparisons test. Data are the means Â± SE of three independent experiments, (n = 6 for A,B).
	Figure 5. Co-expression of HvPIP1s and HvPIP2;8 reduces HvPIP2;8-dependent currents in X. laevis oocytes. (A) Currentâ€“voltage relationships obtained from oocytes either expressing HvPIP2;8 alone, each HvPIP1 alone, or injected with water; each HvPIP1 of the five HvPIP1s did not show ion channel activity when expressed alone. (B) Co-expression of HvPIP2;8 with each HvPIP1 largely inhibited the ion channel activity of HvPIP2;8. (C,D) Box plot summary of the ionic conductance for data shown in (A,B), respectively. Oocytes were injected with 10 ng cRNA of HvPIP2;8, 40 ng cRNA of each HvPIP1 in both the solo- and co-expression analyses. The bath solution included 86.4 mM NaCl, 9.6 mM KCl, 1.8 mM MgCl2, 1.8 mM EGTA, 1.8 mM CaCl2, 10 mM HEPES, and a pH 7.5 with Tris, and therefore the free Ca2+ concentration was 30 ÂµM. Significant differences (p < 0.05) are indicated by different letters using one-way ANOVA with Duncanâ€™s multiple comparisons test. Data are the means Â± SE (n = 8).
	Figure 6. Phosphorylation mimic HvPIP2;8 S285D influences HvPIP2;8-facilitated cation transport. Oocytes were injected with 46 nL water (Control) or with 46 nL water (n = 11) containing 23 ng HvPIP2;8 WT (n = 30) or HvPIP2;8 S285D (n = 40) cRNA. Ionic conductance and osmotic water permeability (Pos) of the cRNA-injected oocytes were determined via the TEVC and the swelling assay, respectively. (A) Na+ conductance relative to H2O-injected control (dotted line). Currents were recorded in â€œNa100â€ (100 mM NaCl; 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, 50 Î¼M CaCl2, 100 mM NaCl or 100 mM KCl, osmolality 220 mosmol Kgâˆ’1 (adjusted with D-mannitol), and pH 8.5). (B) Pos relative to H2O-injected control (dotted line). Data in (A,B) was collected from three different frogs and is shown as the mean Â± SE, where each data point represents an individual oocyte; for each oocyte, both the ionic conductance and Pos were measured; data from (C,D) is from one batch and again for each oocyte both the ionic conductance and Pos were measured. Significant differences (p < 0.05) are indicated by different letters (one-way ANOVA, Fisherâ€™s post-test). (C) Relationships between the mean Pos and mean ionic conductance for HvPIP2;8 WT and HvPIP2;8 S285D with the mean of the controls subtracted. This data is from oocytes from the same three independent frogs as shown in (A,B). (D) Kinase inhibitor H7 influenced the relationship between the ionic conductance and water permeability in HvPIP2;8 S285D-expressing oocytes. Oocytes were either untreated or were pre-treated in a low Na+ Ringer solution that contained 10 ÂµM H7 dihydrochloride (H7) for 2 h before TEVC and the swelling assay. An individual conductance was plotted against the corresponding Pos for each oocyte, and the mean for the water-injected controls is shown (black circle, dotted line). Linear regression of Pos versus ionic conductance was only significant for HvPIP2;8 S285D without H7 treatment (p < 0.005).
	Figure 7. The expression level of the HvPIP2;8 transcripts in a barley cultivar, Haruna-Nijo, detected by qPCR. Five-day-old barley seedlings were prepared by hydroponic culture and further grown on the culture solution with or without NaCl (100 mM or 200 mM) for 1 day or 5 days. Transcript levels of HvPIP2;8 in shoots and roots were investigated by absolute quantification. Absolute amounts of transcripts (copies/Âµg RNA) were displayed. Significant differences (p < 0.05) are indicated by different letters using one-way ANOVA with Duncanâs multiple comparisons test. Data are the means Â± SE, and n = 3.

References [+] :

Amtmann, Multiple inward channels provide flexibility in Na+/K+ discrimination at the plasma membrane of barley suspension culture cells. 1997, Pubmed