A Single-Pore Residue Renders the Arabidopsis Root Anion Channel SLAH2 Highly Nitrate Selective

Abstract

In contrast to animal cells, plants use nitrate as a major source of nitrogen. Following the uptake of nitrate, this major macronutrient is fed into the vasculature for long-distance transport. The Arabidopsis thaliana shoot expresses the anion channel SLOW ANION CHANNEL1 (SLAC1) and its homolog SLAC1 HOMOLOGOUS3 (SLAH3), which prefer nitrate as substrate but cannot exclude chloride ions. By contrast, we identified SLAH2 as a nitrate-specific channel that is impermeable for chloride. To understand the molecular basis for nitrate selection in the SLAH2 channel, SLAC1 and SLAH2 were modeled to the structure of HiTehA, a distantly related bacterial member. Structure-guided site-directed mutations converted SLAC1 into a SLAH2-like nitrate-specific anion channel and vice versa. Our findings indicate that two pore-occluding phenylalanines constrict the pore. The selectivity filter of SLAC/SLAH anion channels is determined by the polarity of pore-lining residues located on alpha helix 3. Changing the polar character of a single amino acid side chain (Ser-228) to a nonpolar residue turned the nitrate-selective SLAH2 into a chloride/nitrate-permeable anion channel. Thus, the molecular basis of the anion specificity of SLAC/SLAH anion channels seems to be determined by the presence and constellation of polar side chains that act in concert with the two pore-occluding phenylalanines.

Figure 1.

Activation of SLAH2 via Interaction with Members of Distinct Protein Kinase Families. (A) and (B) BiFC experiments revealed that SLAH2 physically interacts with CPK21 and CIPK23. SLAH2:YFP^CT was either coexpressed with CPK21ΔEF:YFP^NT (A) (a Ca²⁺-independent and constitutively active CPK21 mutant lacking the EF-hands and the junction domain) or with CIPK23:YFP^NT/CBL1 (B) in oocytes. Representative cells are shown. (C) and (D) Whole-oocyte currents of SLAH2-expressing Xenopus oocytes recorded in the presence or absence of 30 mM NaNO₃⁻. Voltage pulses lasting 20 s were applied ranging from +40 to −180 mV in 20-mV decrements followed by a 3-s voltage pulse to −120 mV. The holding potential was clamped to 0 mV. Coexpression of SLAH2 and CPK21ΔEF (C) or CIPK23/CBL1 (D) in oocytes resulted in macroscopic anion currents in the presence of external NO₃⁻ only. The currents slowly deactivated at negative membrane potentials. Representative cells are shown. (E) SLAH2-mediated currents induced by the coexpression with either CPK21ΔEF or CIPK23/CBL1 did not differ significantly in current amplitudes. The kinase-inactive mutant CPK21ΔEF D204A was not able to elicit SLAH2-mediated currents comparable to those attained with CPK31ΔEF. Activation of SLAH2 by CIPK23 appeared only in the presence of CBL1. However, the coexpression of SLAH2 with the kinase inactive mutant CIPK23 K60N/CBL1 led to no channel activation. The same result was obtained after coexpression of SLAH2 with CIPK24/CBL4 (n ≥ 3, mean ± se). (F) The relative open probability (rel. P_O) of SLAH2 coexpressed with CPK21ΔEF or CIPK23/CBL1 was plotted against the membrane potential. Data points were fitted with a Boltzmann equation (solid lines, n ≥ 4, mean ± sd). [See online article for color version of this figure.]

Figure 2.

NO₃⁻ Dependence of SLAH2 Currents. (A) Steady state currents (I_SS) of SLAH2- and CPK21ΔEF-coexpressing oocytes were normalized to the values at +40 mV in 100 mM NO₃⁻ and plotted against the membrane potential. Currents were measured at the indicated voltages in the presence of the indicated concentrations of external NO₃⁻ concentrations and 3 mM Cl⁻ (n ≥ 5 experiments, mean ± sd). (B) The relative open probability (rel. P_O) of SLAH2 at various NO₃⁻ concentrations plotted against membrane potential. Data points were fitted with a single Boltzmann equation (solid lines, n ≥ 5 experiments, mean ± sd). (C) Reversal potentials V_rev of SLAH2- and CPK21ΔEF-expressing oocytes are shown as a function of the logarithmic external nitrate concentration. As expected for an anion-selective channel, the reversal potential shifted with a slope of 58 mV per decade to more negative values with increasing nitrate concentrations. To avoid loading of the oocytes with nitrate during the measurement, the reversal potentials were recorded in the current-clamp modus (n = 3 experiments, mean ± sd). (D) Relative permeability (rel. permeability) of SLAH2 coexpressed with CPK21ΔEF in Xenopus oocytes (permeability for NO₃⁻ was set to 1). Standard bath solution contained 50 mM of the indicated anion (pH 7.5). The reversal potentials used for the calculation of the relative permeability were recorded in the current-clamp modus (n = 4 experiments, mean ± sd). (E) NO₃⁻ and voltage dependence of steady state currents (I_SS) of SLAH2- and CPK21ΔEF-coexpressing oocytes. Currents were normalized to +40 mV in 100 mM NO₃⁻ + 3 mM Cl⁻ and plotted against the membrane potential. With 10 mM NO₃⁻ in the bath medium SLAH2-mediated currents could be observed, which decreased at negative membrane potentials. The addition of 100 mM Cl⁻ to the bath medium in the presence of 10 mM NO₃⁻ had no influence on the reversal potential (V_rev). By contrast, an increase in the external NO₃⁻ concentration shifted the reversal potential to more negative voltages (n = 4 experiments, mean ± sd). (F) Relative voltage-dependent open probabilities (rel. P_O) of SLAH2- and CPK21-mediated currents under conditions used in Figure 2E. Rel. P_O was calculated from a −120-mV voltage pulse following the test pulses in the voltage range +60 to −200 mV in 20-mV decrements. Data points were fitted by a Boltzmann equation (continuous line; n = 4 experiments, mean ± sd).

Figure 3.

Anion Selectivity of SLAH2. Whole-oocyte currents of oocytes coexpressing SLAH2, CIPK23, and CBL1 in standard medium containing 30 mM NO₃⁻. The experiment was performed at two different holding potentials using the standard voltage protocol. (A) When the oocytes were clamped to 0 mV, single voltage pulses were applied starting from +60 to −200 mV in 20-mV decrements. Representative cells are shown. (B) When the oocytes were clamped to −100 mV, the voltage pulses increased from −200 to +60 mV in 20-mV steps. Representative cells are shown. (C) Steady state currents (I_SS) of SLAH2 and CIPK23/CBL1 coexpressing oocytes measured as in (A) (V_hold = 0 mV pos > neg) or (B) (V_hold = −100 mV neg > pos) (n ≥ 5 experiments, mean ± sd).

Figure 4.

Structural Model of SLAC1 and SLAH2. (A) and (B) Ribbon plot of the homology models of SLAC1 (cyan) and SLAH2 (magenta). (A) Channel seen from the extracellular side, with the side chains of the conserved Phe-450/402 and Phe-276/231 occluding the pore. (B) Same as in (A) but shown as a side view.

Figure 5.

Mutations in TM3 Influence Ion Selectivity of SLAC1 and SLAH2. Instantaneous currents (I_inst) of Xenopus oocytes in standard buffers containing different anions (gluconate, chloride, or nitrate) are plotted against the applied voltage. (A) and (C) SLAH2 wild type and the mutant SLAH2C1(271-281). (B) and (D) SLAC1 wild type and the corresponding mutant. The channel mutants and kinases expressed in the oocytes are indicated in the figure (n ≥ 3 experiments, mean ± se).

Figure 6.

Structural Differences of Transmembrane Helix 3 Determine the Selectivity Differences between SLAC1 and SLAH2. (A) to (C) Structural differences in the pore region of SLAC1 and SLAH2. Close-up view of TM3 of SLAC1 (A), an overlay of SLAC1 and SLAH2 (B), and SLAH2 (C) with the side chains and TM4 of SLAC1 (carbon atoms in cyan) and SLAH2 (carbon atoms in magenta). Pore lining residues are facing left. The position of pore-blocking Phe-450 and Phe-402 on TM9 is shown. (D) and (E) Instantaneous currents (I_inst) of Xenopus oocytes coexpressing the mutants SLAH2 S228V (C) or SLAC1 V273S (D) with CIPK23/CBL1 in standard buffers containing different anions (gluconate, chloride, or nitrate) are plotted against the applied voltage (n ≥ 4 experiments, mean ± se). (F) Chord conductance at +40 and −120 mV in standard solutions containing 100 mM Cl⁻. The chord conductance was calculated from the instantaneous currents in Cl⁻- as well as in NO₃⁻-containing buffers from oocytes coexpressing SLAH2 or SLAC1 wild type, or the different channel mutants, with CIPK23 and CBL1. The chord conductance was normalized to 100 mM NO₃⁻ (dashed line) (n ≥ 3 experiments, mean ± se).