XB-ART-51415Mol Plant. March 7, 2016; 9 (3): 428-436.
Venus Flytrap HKT1-Type Channel Provides for Prey Sodium Uptake into Carnivorous Plant Without Conflicting with Electrical Excitability.
The animal diet of the carnivorous Venus flytrap, Dionaea muscipula, contains a sodium load that enters the capture organ via an HKT1-type sodium channel, expressed in special epithelia cells on the inner trap lobe surface. DmHKT1 expression and sodium uptake activity is induced upon prey contact. Here, we analyzed the HKT1 properties required for prey sodium osmolyte management of carnivorous Dionaea. Analyses were based on homology modeling, generation of model-derived point mutants, and their functional testing in Xenopus oocytes. We showed that the wild-type HKT1 and its Na(+)- and K(+)-permeable mutants function as ion channels rather than K(+) transporters driven by proton or sodium gradients. These structural and biophysical features of a high-capacity, Na(+)-selective ion channel enable Dionaea glands to manage prey-derived sodium loads without confounding the action potential-based information management of the flytrap.
PubMed ID: 26455461
PMC ID: PMC4791408
Article link: Mol Plant.
Genes referenced: pigy
Article Images: [+] show captions
|Figure 1. In Vivo Measurement of Electrical Excitability of the Venus Flytrap.(A) Surface potential of Venus flytrap revealed AP firing in response to touch and 3% NaCl treatment.(B) Membrane voltage recording of impaled Dionaea glands exposed to 3% NaCl. This near seawater concentration evoked spontaneous electrical activity with up to 20 APs/min.(C) Membrane voltage recording of impaled Dionaea glands in the absence of Na+ (black) and presence of 510 mM Na+ (red). Mechanical stimulation of a trigger hair resulted in firing of APs, which did not differ clearly in amplitude and profile. Representative APs are shown.|
|Figure 2. DmHKT1-Driven Na+ Flux Is Not Coupled to Other Ions.(A) In MIFE experiments with unstimulated and COR-treated Dionaea traps, net Na+ and K+ influxes were measured simultaneously under the indicated external Na+ concentrations. Unstimulated traps under these conditions showed only a small Na+ uptake of 310 ± 415 nmol m−2 s−1. Only stimulated traps revealed a clear Na+ influx, even under low external sodium concentrations, which increases up to 1683 ± 218 nmol m−2 s−1 with 50 mM external NaCl. K+ influx was not significantly affected by a change in the external Na+ concentration in either unstimulated or COR-treated traps (n ≥ 7, mean ± SD; P > 0.5, one-way ANOVA).(B) Na+ influx in Dionaea glands is H+-independent. Normalized Na+ fluxes from COR-stimulated Venus flytraps were measured in low (0.1 mM; left) and high (10 mM; middle) NaCl concentrations at a pH of 4. Under low external NaCl, the Na+ influx was only 3.7% ± 5.8% of that in the high external sodium. Increasing the external pH from 4 to 8 (without changing Na+ concentration; right) did not significantly influence the Na+ flux (109% ± 19%) (n ≥ 7, mean ± SD; P > 0.5 by one-way ANOVA).(C) For different monovalent cations at a concentration of 100 mM, the chord conductance was calculated and plotted against the applied voltage range of −40 to −200 mV (n = 4, mean ± SD). DmHKT1 was only permeable for sodium.(D) DmHKT1-mediated inward currents in 5 mM NaCl of a representative Xenopus oocyte when the membrane was clamped to −140 mV. The current amplitude did not change significantly by applying Ca2+ concentrations of 0.1, 1, and 10 mM (as indicated).|
|Figure 3. Altered Conductance and Affinity in the DmHKT1 S84G Mutant.(A) Steady-state currents (Iss) of DmHKT1 wild-type (WT) and S84G-expressing oocytes measured at −140 mV and in 100 mM of the indicated cations (n = 4, mean ± SD).(B) Steady-state currents (Iss) of DmHKT1 S84G at −140 mV were monitored in and plotted against increasing K+ concentrations up to 100 mM (please note different scaling). The current response reached a maximum at 50 mM KCl and with higher concentrations, the K+ inward currents declined. Currents up to 50 mM K+ could be fitted with the Michaelis–Menten equation revealing a Km value of 9.01 ± 1.99 mM K+ (n = 5, mean ± SD).(C) By applying 10 mM NaCl, 10 mM KCl, and a combination of 10 mM of each cation at −140 mV, S84G-mediated currents under combined ion conditions were the sum of the current responses to each cation alone (n = 5, mean ± SD).(D) Steady-state currents (Iss) of the WT DmHKT1 and the mutant S84G were monitored in bath solutions containing 10 mM KCl and a stepwise increase in sodium concentrations. WT-mediated currents could be fitted with the Michaelis–Menten equation, whereas the current response of S84G proceeded in a linear-dependent manner (n ≥ 4, mean ± SD).(E) In inverse experiments, steady-state currents (Iss) of the WT and the mutant S84G were measured in 10 mM NaCl and a stepwise increase in potassium concentrations (n ≥ 4, mean ± SD). The resulting currents showed behavior opposite to that shown in (D).(F) The AMFE of S84G-expressing cells (n = 4, mean ± SD) analyzed in increased Na+ concentrations, supplemented with K+ to a final concentration of 50 mM. To maintain the ionic strength, 50 mM LiCl was used. The steady-state currents (Iss) at −140 mV were plotted against the sodium concentration.|
|Figure 4. Homology Modeling of the DmHKT1 Pore.(A) DmHKT1 channel (ribbon plot in cyan) embedded in an artificial phosphatidylethanolamine (POPE) membrane bilayer (lipid molecules shown as stick representation). The model of DmHKT1 was placed in the bilayer using the ProBML web server (http://compbio.clemson.com/problm_webserver). The thickness of the membrane bilayer and the distance between the potassium ion (magenta sphere) in the selectivity filter and the upper boundary of the membrane were measured with Quanta2008. The insert shows the Km dependency of DmHKT1 S84G on clamped voltage. Data were fitted with an exponential function (Km = Km0 exp[δzFVm/RT]). Since zF/RT = 0.0417 mV−1, the resulting parameters were Km0 = 139.46 ± 11.54 mM and δ = 0.370 ± 0.012 (n = 6). Currents and voltages were recorded with standard bath solution containing indicated K+ concentrations.(B) 3D homology model of the D. muscipula ion channel DmHKT1 shown as ribbon plot with the chain colored from blue (N terminus) to red (C terminus). The pore and metal-binding site are indicated.(C) Magnification of the four pore-lining loops of wild-type DmHKT1 with the carbon atoms colored blue (loop 1, amino acids [aa] Thr81 to Ser86), green (loop 2, aa Ser235 to Thr240), yellow (loop 3, aa Arg359 to Ser364), and red (loop 4, aa Gly464 to Ser469). The hydroxyl group of serine 84 is located inside the pore lumen and participates in the coordination of the sodium ion (magenta sphere). Stippled lines indicate the metal ion coordination by the surrounding carbonyl groups of Val82, Ser236, Val237, His360, Thr361, Asn465, and Val466, and the hydroxyl group of Ser84. The distance between the carbonyl group of Val82 and the metal ion exceeds 3.2 Å, and therefore may not contribute to metal coordination (black stippled line).(D) Magnification of the DmHKT1 variant with a glycine residue occupying position 84 (S84G). The exchange of serine 84 in the first pore-lining loop by glycine results in altered backbone conformation of the preceding amino acid residues Ser83 and Val82 (see also Supplemental Figure 3), thereby reorienting their carbonyl groups toward the metal coordination site in the pore. This conformation is also found in potassium-conducting bacterial ion channels of this channel family, e.g. TrkH and KtrB, suggesting that pore loops containing a central glycine residue can adopt favorable backbone conformations for enhanced potassium binding.(E) Magnification of a mutant ion channel DmHKT1 with alanine at position 84 (S84A). The metal coordination is supposedly similar to wild-type DmHKT1 carrying a serine residue at this position. However, the missing serine hydroxyl group, hence the lack of one metal coordination site, impairs the Na+–K+ discrimination.(F) Steady-state currents (Iss) of DmHKT1 S84G (black) and DmHKT1 S84A expressing oocytes at −140 mV and in 100 mM of the indicated cations (n = 4, mean ± SD).|