Scherzer S et al. (2015), Calcium sensor kinase activates potassium uptak...

XB-ART-50753

Proc Natl Acad Sci U S A 2015 Jun 09;11223:7309-14. doi: 10.1073/pnas.1507810112.

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Calcium sensor kinase activates potassium uptake systems in gland cells of Venus flytraps.

Scherzer S, Böhm J, Krol E, Shabala L, Kreuzer I, Larisch C, Bemm F, Al-Rasheid KA, Shabala S, Rennenberg H, Neher E, Hedrich R.

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The Darwin plant Dionaea muscipula is able to grow on mineral-poor soil, because it gains essential nutrients from captured animal prey. Given that no nutrients remain in the trap when it opens after the consumption of an animal meal, we here asked the question of how Dionaea sequesters prey-derived potassium. We show that prey capture triggers expression of a K(+) uptake system in the Venus flytrap. In search of K(+) transporters endowed with adequate properties for this role, we screened a Dionaea expressed sequence tag (EST) database and identified DmKT1 and DmHAK5 as candidates. On insect and touch hormone stimulation, the number of transcripts of these transporters increased in flytraps. After cRNA injection of K(+)-transporter genes into Xenopus oocytes, however, both putative K(+) transporters remained silent. Assuming that calcium sensor kinases are regulating Arabidopsis K(+) transporter 1 (AKT1), we coexpressed the putative K(+) transporters with a large set of kinases and identified the CBL9-CIPK23 pair as the major activating complex for both transporters in Dionaea K(+) uptake. DmKT1 was found to be a K(+)-selective channel of voltage-dependent high capacity and low affinity, whereas DmHAK5 was identified as the first, to our knowledge, proton-driven, high-affinity potassium transporter with weak selectivity. When the Venus flytrap is processing its prey, the gland cell membrane potential is maintained around -120 mV, and the apoplast is acidified to pH 3. These conditions in the green stomach formed by the closed flytrap allow DmKT1 and DmHAK5 to acquire prey-derived K(+), reducing its concentration from millimolar levels down to trace levels.

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Species referenced: Xenopus
Genes referenced: akt1 pigy

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	Fig. 2. Transport activation of DmHAK5. (A) Normalized current response recorded at −140 mV of Xenopus oocytes injected with H2O, cRNA of DmHAK5, or cRNA of DmHAK5 supplemented with CBL9/CIPK23. Macroscopic potassium currents were only detectable in DmHAK5/CBL9/CIPK23-coexpressing oocytes in solutions containing 2 mM potassium (n ≥ 6; mean ± SD). (B) Typical whole-oocyte currents recorded at −60 mV of an H2O-injected (black) and a DmHAK5/CBL9/CIPK23-expressing (red) Xenopus oocyte. Addition of 2 mM K+ resulted in macroscopic inward currents of ∼400 nA only with DmHAK5-expressing oocytes. The dotted line represents zero current.
	Fig. 3. Selectivity and pH dependency of DmKT1. (A) Currents recorded at −140 mV of DmKT1/CBL9/CIPK23-expressing oocytes in the presence of bath solutions containing various monovalent cations (each 100 mM). Large cation influx was only obtained with potassium in the external bath solution (n = 4; mean ± SD). (B) Inward K+ currents elicited by a DmKT1/CBL9/CIPK23-expressing Xenopus oocyte markedly increased on acidification of the external solution (Vm = −100 mV). Exchanging 100 mM external Li+ for 100 mM K+ at pH 6 resulted in potassium influx, which was enhanced by increasing the external H+ concentration. The dotted line represents zero currents. (C) Normalized currents of DmKT1/CBL9/CIPK23-expressing Xenopus oocytes at −140 mV. Stepwise acidification from pH 6 to 3 increased the potassium currents through DmKT1 (n = 6; mean ± SD). (D) The relative open probability (rel. PO) of DmKT1-expressing oocytes at the indicated H+ concentrations was plotted against the applied test voltage. Note the prominent positive shift of the half-maximal activation potential (V1/2) with increasing acidification. The data points were fitted with a Boltzmann function (solid lines; n = 6; mean ± SD).
	Fig. 4. Selectivity and pH dependency of DmHAK5. Xenopus oocytes were either injected with H2O (control) or cRNA of DmHAK5/CBL9/CIPK23 (DmHAK5). (A) Representative current recordings at −60 mV of a control (black) and a DmHAK5-expressing (red) Xenopus oocyte in response to 2 mM Li+, Na+, K+, Rb+, Cs+, NH4+, or NMDG+ (as indicated using sorbitol-based solutions). Both K+ and Rb+ resulted in macroscopic inward currents with DmHAK5-expressing oocytes. Application of NH4+ and Cs+ induced weaker DmHAK5-mediated inward currents. The dotted line represents zero current. (B) Average results from experiments as shown in A, except that the holding potential was −140 mV (n = 7; mean ± SD). (C) Normalized steady-state currents of Xenopus oocytes injected with DmHAK5 were recorded at −140 mV in response to sorbitol- or Na+-based solution (200 mOsm/kg). Addition of 2 mM K+ resulted in identical K+ influx irrespective from the presence or absence of sodium in the bath solution (n = 7; mean ± SD). (D) ISS recorded at −140 mV with oocytes injected with either H2O (gray bars) or DmHAK5 (red bars) in the presence of 100 mM K+ at different pH values (as indicated; n = 5; mean ± SD). Compared with control oocytes, K+ currents increased at pH values ≤5.
	Fig. 5. Biophysical analysis of DmKT1- and DmHAK5-mediated K+ currents at varying extracellular K+ concentrations. (A) Dose–response curve of DmKT1/CBL9/CIPK23-expressing Xenopus oocytes at −140 mV. The ionic strength of the solutions with varying K+ concentrations was adjusted to 100 mM with Li+. The saturation curve could be best fitted with a Michaelis–Menten equation (solid line), documenting a Km value of 108.29 ± 7.34 mM for DmKT1 (n = 5; mean ± SD). (B) Dose–response curve of DmHAK5/CBL9/CIPK23-expressing Xenopus oocytes clamped to a membrane potential of −140 mV. The Michaelis–Menten fit revealed a Km value of 127.38 ± 2.77 µM for DmHAK5 (n = 6; mean ± SD). (C) GK/V relationship of DmKT1-expressing oocytes in a K+ concentration range from 100 to 5 mM. Data points were normalized to GK at −140 mV in 100 mM K+ and fitted with a Boltzmann function. Decreasing the potassium concentration leads to a reduction in the cord conductance but not a change of the voltage dependence of the relative open probability (see also Fig. S5B) (n = 5; mean ± SD). (D) The relative maximal cord conductance was calculated with the equation: GK−max = GK−max(X mM)/GK−max (100 mM). Data points were plotted against the applied K+ concentration and fitted with a Michaelis–Menten equation. Note that GK−max increased with increasing potassium concentrations with a half-maximal activity (K0.5) of 34 mM (n = 5 ± SD).

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