Arabidopsis HAK5 under low K+ availability operates as PMF powered high-affinity K+ transporter

The study addresses the long-standing question of how Arabidopsis thaliana HAK5 mediates high-affinity potassium uptake under low external K+ and whether it functions as a proton-motive-force (PMF)-driven K+/H+ symporter. Potassium is a major inorganic cation essential for plant turgor and membrane potential, with root cytosolic [K+] maintained near 100 mM while rhizosphere [K+] can fall to low micromolar levels, creating steep gradients. Classical tracer work described biphasic root K+ uptake with a high-affinity system (~20 µM) and a low-affinity system (200 µM–2 mM). The inward-rectifying K+ channel AKT1 supports uptake in the low-affinity range and is activated by the CBL1/CIPK23 kinase complex; however, AKT1 does not recognize K+ in the low micromolar range. HAK5, a KUP/HAK/KT family transporter, has been genetically implicated in high-affinity K+ uptake, but direct electrophysiological evidence for its transport mechanism has been lacking and NH4+ confounds tracer-based assays. This work aims to directly quantify AtHAK5-mediated currents, define its ion coupling and gating by voltage and pH, and pinpoint structural determinants of K+ sensing.

Prior studies established biphasic K+ uptake in roots and implicated AKT1 channels and HAK/KUP/KT transporters in low- and high-affinity ranges, respectively. Loss of AKT1 impairs uptake below ~1 mM K+. Tracer Rb+ assays and NH4+ competition were used to estimate HAK5 contributions, but these approaches are confounded because some K+ channels poorly conduct Rb+ and NH4+ is a substrate for both K+ channels and ammonium transporters. Both AKT1 and HAK5 are activated by the CBL1/CIPK23 kinase pathway, which is transcriptionally regulated by STOP1 under low K+. Structural work on the bacterial KUP-family transporter KimA revealed K+-binding sites and proposed K+/H+ symport. High-affinity NH4+ AMT transporters complicate attributing NH4+ fluxes to HAK5 in planta. Collectively, the literature motivated direct current measurements and mechanistic dissection of HAK5.

- Expression system: Xenopus laevis oocytes heterologously expressing Arabidopsis AtHAK5, with or without co-expression of CBL1/CIPK23 to activate the transporter. Control oocytes were water-injected. - Electrophysiology: Two-electrode voltage clamp (TEVC). Membrane potentials stepped typically from -60 to -140 mV; standard assays at -120 mV. External solutions varied in K+, Rb+, Cs+, Na+, Li+, NH4+ and pH (4.0–8.5). Leak/background currents from control oocytes were subtracted. - Ion-selective measurements: Simultaneous monitoring of bath [K+] with K+-selective electrodes and pH with H+-selective electrodes during TEVC protocols to confirm applied concentrations and pH. - pH coupling assays: N-terminal pHluorin2 fused to HAK5 to report cytosolic pH changes during K+-evoked currents; cytosolic acidification with sodium acetate to collapse ΔpH and test PMF dependence. - Dose–response analyses: Peak and steady-state currents quantified across K+ ranges (10 µM–2 mM for WT; up to 100 mM for Y450A) and NH4+ ranges (10–2000 µM). Fits used Michaelis–Menten for cations and Hill functions for H+ dependence. Voltage dependence of Imax and Km for K+ and H+ determined. - Inactivation kinetics: 120 s pulses at -120 mV after K+ application (10 µM–2 mM; varying pH) to quantify time-dependent current decay (percentage inactivation = (ΔIpeak − ΔIss)/ΔIpeak × 100). - Structure-function: Homology model of AtHAK5 transmembrane core (Q64–R541) built on KimA (PDB 6S3K) to identify putative K+-coordination residues. Site-directed mutagenesis of D72, D201, E312 (predicted binding residues) and Y450 (equivalent to KimA Y377). Functional assessment by TEVC. - Kinetic modeling: Four-state scheme derived from a six-state K+/H+ symport model to capture voltage dependence of K+ vs H+ binding and transport. Analytical solutions (King-Altman method) fitted to experimental data; parameter adjustments used to simulate effects of Y450A (altered K+ binding constant). - Experimental details: Oocyte preparation, cloning via uracil-excision-based methods, cRNA synthesis, composition of perfusion buffers, calibration of ion-selective electrodes, and data acquisition/analysis software are described in Methods.

- Activation conditions and affinity: - AtHAK5 requires CBL1/CIPK23 for robust activity; co-expression increased inward K+ currents ~10-fold over AtHAK5 alone. - No HAK5 current at sufficient K+ (2 mM) despite AKT1 showing strong inward currents at 2 mM K+. - Low K+ (10–200 µM) activated HAK5; peak currents vs [K+] fit with Michaelis–Menten yielded Km(K+) = 23.56 ± 1.3 µM at pH 4.5. - Steady-state currents exhibited a bell-shaped dependence on [K+], peaking near ~28 µM, coincident with Km. - Substrate selectivity: - Rb+ supported currents similar to K+ (Km(Rb+) = 22.96 ± 7.1 µM); Cs+ currents smaller (~51% of K+) with Km(Cs+) = 18.90 ± 12.9 µM; Li+ and Na+ did not elicit inward currents. - NH4+ is also transported: currents ~37.6 ± 9.6% smaller than K+ with lower affinity (Km(NH4+) = 69.36 ± 17.9 µM). ~5× higher NH4+ needed to match currents from 10 µM K+. - At 1 mM NH4+, adding 10–100 µM K+ did not further increase current, indicating NH4+ outcompetes K+ binding at high NH4+. - Proton coupling and pH dependence: - pHluorin:HAK5 recordings showed K+-evoked inward currents tightly coupled to cytosolic acidification, reverting on washout, consistent with K+/H+ symport. - Decreasing external pH (increasing [H+]) increased currents; half-maximal activity at [H+] = 4.81 ± 1.3 µM (pH dependent) with 200 µM K+. With 20 µM K+, half-max at 3.15 ± 0.8 µM H+. - Collapsing ΔpH by cytosolic acidification (sodium acetate) markedly reduced K+-evoked currents at fixed external pH, demonstrating PMF drives transport. - Voltage dependence: - Imax up to 417 ± 65 nA at -140 mV; currents decreased with depolarization, ceasing near -40 mV. Km(K+) weakly voltage dependent. - Km(H+) decreased (higher apparent H+ affinity) at more negative potentials; a +30 mV depolarization (-120 to -90 mV) increased Km(H+) by ~20 µM, indicating proton binding senses the electric field while K+ binding does not. - Inactivation kinetics: - At 2 mM K+, currents peaked within ~2 s then inactivated to near zero by 120 s; at 200 µM K+ similar but less pronounced; at 10–20 µM K+, currents reached and maintained steady-state without inactivation. - Inactivation increased as external pH rose (lower [H+]); e.g., at 20 µM K+, no inactivation at pH 4.5 but strong inactivation at pH 6.5. - Structure-function and mutagenesis: - Homology modeling (KimA-based) identified a well-coordinated upper K+ site (D72, T75, S76, Y79, T205, S209, Y450) and a lower cavity (D72, D201, T205, T309) in AtHAK5. - D72A, D201A, or E312A rendered HAK5 electrically silent, consistent with essential roles in ion binding. - Y450A preserved function but drastically shifted K+ affinity: Km(K+) = 12.54 ± 1.3 mM (≈500-fold decrease in affinity). Rb+ Km ≈ 22.8 ± 1.3 mM; Cs+ Km ≈ 78.6 ± 10.8 mM; NH4+ Km ≈ 309.6 ± 88.2 mM. Y450A currents lacked inactivation at low mM K+ but showed pronounced inactivation at higher [K+]. - Y450A increased apparent H+ affinity: Km(H+) shifted from 4.81 ± 1.3 µM (WT) to 0.36 ± 0.02 µM. - Four-state kinetic model reproduced experimental voltage and ligand dependences and Y450A effects by reducing the external K+ binding rate constant (k23), supporting interplay between K+ and H+ binding. - Physiological implication: - Under low micromolar K+, AKT1-mediated uptake ceases while HAK5 activates to maintain K+ uptake using PMF; at higher K+, HAK5 inactivates, preventing unnecessary PMF dissipation and depolarization, allowing AKT1 to assume uptake.

Direct TEVC measurements in a plant-background–free system establish AtHAK5 as a CIPK23/CBL1-activated, PMF-powered high-affinity K+/H+ symporter. The transporter switches on at low external K+ (peak around ~20–30 µM), shows strong dependence on external H+ and membrane hyperpolarization, and exhibits substrate selectivity consistent with K+ preference over NH4+. The coupling to H+ allows theoretical K+ accumulation against extreme gradients, extending uptake capacity well below the range supported by AKT1 channels. Time-dependent inactivation at higher K+ and higher pH suggests an intrinsic K+-sensing mechanism that downregulates HAK5 when soil K+ is sufficient, thereby conserving energy and stabilizing membrane potential and cytosolic pH. Structural modeling and mutational analysis identify essential acidic residues (D72, D201, E312) for transport and pinpoint Y450 as a critical determinant of K+ sensing and the coordination between K+ and H+ binding, aligning with KimA structural insights. The kinetic model integrates these observations, attributing voltage sensitivity primarily to proton-binding and to the voltage-dependent conformational step, while K+ binding is largely voltage-insensitive. Collectively, the results resolve the debated mechanism by providing direct current evidence for PMF-coupled high-affinity K+ transport by HAK5 and clarify its role relative to AKT1 in K+ homeostasis under fluctuating soil K+.

This work provides direct electrophysiological evidence that Arabidopsis HAK5 is a high-affinity, PMF-driven K+/H+ symporter activated by the CBL1/CIPK23 kinase complex. HAK5 activates under low external K+ and inactivates as K+ rises, integrating transport with sensing to optimize energy use and membrane excitability, thereby complementing AKT1 channel function. Structural and kinetic analyses identify critical residues for ion binding and demonstrate that Y450 governs K+ affinity and coordinates K+/H+ coupling. These insights inform strategies to enhance crop K-use efficiency and resilience on K-poor soils. Future research should test HAK5’s putative transceptor role by probing downstream signaling and transcriptomic responses in hak5 mutants under K+ starvation and further elucidate the molecular basis of inactivation and its regulation in planta.

- Experiments were conducted in Xenopus oocytes; while plant background–free and robust, heterologous systems may not capture all regulatory interactions present in planta. - Direct downstream signaling roles of HAK5 (transceptor function) remain unresolved; the link between HAK5-mediated sensing, transport regulation, and gene expression is still scant and proposed as future work. - The homology model covers only the transmembrane core (Q64–R541); low sequence homology prevented modeling of terminal cytosolic domains, potentially omitting regulatory elements. - The precise molecular mechanism of K+-dependent inactivation and its modulation in planta is not fully elucidated.