Characterization of two HKT1 homologues from Eucalyptus camaldulensis that display intrinsic osmosensing capability

Abstract

Plants have multiple potassium (K(+)) uptake and efflux mechanisms that are expressed throughout plant tissues to fulfill different physiological functions. Several different classes of K(+) channels and carriers have been identified at the molecular level in plants. K(+) transporters of the HKT1 superfamily have been cloned from wheat (Triticum aestivum), Arabidopsis, and Eucalyptus camaldulensis. The functional characteristics as well as the primary structure of these transporters are diverse with orthologues found in bacterial and fungal genomes. In this report, we provide a detailed characterization of the functional characteristics, as expressed in Xenopus laevis oocytes, of two cDNAs isolated from E. camaldulensis that encode proteins belonging to the HKT1 superfamily of K(+)/Na(+) transporters. The transport of K(+) in EcHKT-expressing oocytes is enhanced by Na(+), but K(+) was also transported in the absence of Na(+). Na(+) is transported in the absence of K(+) as has been demonstrated for HKT1 and AtHKT1. Overall, the E. camaldulensis transporters show some similarities and differences in ionic selectivity to HKT1 and AtHKT1. One striking difference between HKT1 and EcHKT is the sensitivity to changes in the external osmolarity of the solution. Hypotonic solutions increased EcHKT induced currents in oocytes by 100% as compared with no increased current in HKT1 expressing or uninjected oocytes. These osmotically sensitive currents were not enhanced by voltage and may mediate water flux. The physiological function of these osmotically induced increases in currents may be related to the ecological niches that E. camaldulensis inhabits, which are periodically flooded. Therefore, the osmosensing function of EcHKT may provide this species with a competitive advantage in maintaining K(+) homeostasis under certain conditions.

Figure 1

Effects of 1 mm Na⁺, Rb⁺, Li⁺, and Cs⁺ on K⁺ currents in EcHKT1- and EcHKT2-expressing and uninjected Xenopus laevis oocytes. Current traces recorded at −120 mV from an uninjected (A), EcHKT1-expressing (B), and EcHKT2-expressing (C) oocyte. Traces with K⁺ and Na⁺ in bath have been labeled and the rest are traces with Rb⁺, Li⁺, and Cs⁺ that correspond to histograms. Current changes (D and E) in EcHKT1(n = 4 oocytes), EcHKT2 (n = 4), and uninjected oocytes (n = 4) measured in the presence of 1 mm X ion plus 10 mm K⁺ in bath solution. Current difference (Δ current) is the difference between the currents recorded at 0 mm monovalent cations and 1 mm X ion/10 mm K⁺.

Figure 2

Effects of 1 mm Na⁺ on K⁺, Rb⁺, Li⁺, and Cs⁺ in EcHKT1- and EcHKT2-expressing and uninjected X. laevis oocytes. Current traces recorded at −120 mV from an uninjected (A), EcHKT1-expressing (B), and EcHKT2-expressing (C) oocyte. Traces with K⁺ and Na⁺ in bath have been labeled and the rest are traces with Rb⁺, Li⁺, and Cs⁺ that correspond to histograms. Current changes (D and E) measured in EcHKT1(n = 4 oocytes), EcHKT2 (n = 4), and uninjected oocytes (n = 4) in the presence of 1 mm Na⁺ plus 10 mm X ion in bath solution. Current difference (Δ current) is the difference between the currents recorded at 0 mm monovalent cations and 1 mm Na⁺/10 mm X ion.

Figure 3

Effects of bath solutions containing the single monovalent cations Na⁺, Rb⁺, Li⁺, and Cs⁺ on EcHKT1- and EcHKT2-expressing and uninjected X. laevis oocytes. Current traces recorded at −120 mV from an uninjected (A), EcHKT1-expressing (B), and EcHKT2-expressing (C) oocyte. Traces with K⁺ and Na⁺ in bath have been labeled and the rest are traces with Rb⁺, Li⁺, and Cs⁺ that correspond to histograms. Current changes (D and E) measured in EcHKT1 (n = 4 oocytes), EcHKT2 (n = 4), and uninjected oocytes (n = 4) were measured in the presence of single monovalent cation ion (10 mm) in bath solution. Current difference (Δ current) is the difference between the currents recorded at 0 mm monovalent cations and 10 mm cation ion.

Figure 4

Kinetics of inward currents mediated by EcHKT1 or EcHKT2 as a function of external Na⁺ concentration. A, The relationship between Na⁺ concentration and current at 1 mm external K⁺ with curves fitted to the Michaelis-Menten equation. B, The voltage dependence at 1 mm K⁺.

Figure 5

Current-voltage (I-V) relationship in EcHKT1-expressing (n = 6), EcHKT2-expressing (n = 5), and EcHKT1/EcHKT2 co-expressing oocytes (n = 8). Solutions contained 1.8 mm CaCl₂, 10 mm MES[2-(N-morpholino)-ethanesulfonic acid]-Tris, 200 mm sorbitol, 1 mm Na⁺, and 10 mm K⁺ (pH 6.5).

Figure 6

Hypotonic solutions induced increased inward currents in EcHKT1- and EcHKT2-expressing oocytes. Each oocyte tested was bathed in solution containing 200 mm and then the chamber was perfused with solution containing 50 mm sorbitol. Solutions also included 1.8 mm CaCl₂, 10 mm MES-Tris, 1 mm Na⁺, 10 mm K⁺ (pH 6.5), and uninjected oocyte (A), EcHKT1-expressing oocyte (B), and EcHKT2-expressing (C) oocyte. Holding potential was −40 mV and the current traces corresponded to the membrane potentials of −140 mV to +40 mV with 20-mV increments.

Figure 7

I-V relationships in isoosmotic (200 mm sorbitol), hypotonic (50 mm sorbitol), or hypertonic (600 mm sorbitol) solutions. I-V curves from oocytes expressing EcHKT1 (A; n = 6), EcHKT2 (B; n = 8), EcHKT1 and EcHKT2 (C; n = 7), and HKT1 (D; n = 7). Solutions also contained 1.8 mm CaCl₂, 10 mm MES-Tris, 200 mm sorbitol, 1 mm Na⁺, and 10 mm K⁺ (pH 6.5).

Figure 8

Effects of divalent cations on EcHKT1- or EcHKT2-mediated inward currents. Currents at −120 mV and 1 mm Na⁺/10 mm K⁺ without divalent cations were used to normalize the currents at −120 mV measured at 1 mm Na⁺/10 mm K⁺ with 2 mm Ca²⁺, Mg²⁺, and Ba² (EcHKT1, n = 4; EcHKT2, n = 6).