HKT1 Mediates Sodium Uniport in Roots. Pitfalls in the Expression of HKT1 in Yeast

Abstract

The function of HKT1 in roots is controversial. We tackled this controversy by studying Na+ uptake in barley (Hordeum vulgare) roots, cloning the HvHKT1 gene, and expressing the HvHKT1 cDNA in yeast (Saccharomyces cerevisiae) cells. High-affinity Na+ uptake was not detected in plants growing at high K+ but appeared soon after exposing the plants to a K+-free medium. It was a uniport, insensitive to external K+ at the beginning of K+ starvation and inhibitable by K+ several hours later. The expression of HvHKT1 in yeast was Na+ (or K+) uniport, Na+-K+ symport, or a mix of both, depending on the construct from which the transporter was expressed. The Na+ uniport function was insensitive to external K+ and mimicked the Na+ uptake carried out by the roots at the beginning of K+ starvation. The K+ uniport function only took place in yeast cells that were completely K+ starved and disappeared when internal K+ increased, which makes it unlikely that HvHKT1 mediates K+ uptake in roots. Mutation of the first in-frame AUG codon of HvHKT1 to CUC changed the uniport function into symport. The expression of the symport from either mutants or constructs keeping the first in-frame AUG took place only in K+-starved cells, while the uniport was expressed in all conditions. We discuss here that the symport occurs only in heterologous expression. It is most likely related to the K+ inhibitable Na+ uptake process of roots that heterologous systems fail to reproduce.

The Rice Monovalent Cation Transporter OsHKT2;4: Revisited Ionic Selectivity

“…5C). This result, indicating an increased Na + transport activity in OsHKT2;4 in low K + and high Na + conditions, is consistent with what was suggested by the reciprocal experiment described by Figure 4, E and F. It should be noted that HKT2;1-type transporters from different cereals have been proposed to be able to mediate Na + -K + symport when expressed in yeast and/or Xenopus oocytes (Rubio et al, 1995;Haro et al, 2005;Jabnoune et al, 2009;Mian et al, 2011). Within the framework of the hypothesis that OsHKT2;4 could also mediate Na + -K + symport activity with variable stoichiometry, as proposed for HvHKT2;1 transporter when expressed in oocytes (Mian et al, 2011), a shift in reversal potential of currents of 31 mV per 10-fold change in ionic activity is very close to the expected shift in a symport of monovalent cations with a 1:1 stoichiometry (i.e.…”

“…Apart from OsHKT2;4, all the members of subfamily 2 transporters that have been characterized so far are close homologs of TaHKT2;1 Hauser and Horie, 2010), the first subfamily 2 transporter characterized (Rubio et al, 1995). Expressed in heterologous systems, these HKT2;1 subgroup transporters display a complex behavior compared with the transporters from subfamily 1, since they are endowed with different conduction modes (Rubio et al, 1995;Gassmann et al, 1996;Haro et al, 2005;Jabnoune et al, 2009). They work as Na + -K + symporters at low Na + and K + concentrations, when Na + and K + are in the submillimolar range, and eventually at higher (millimolar) concentrations when Na + and K + concentrations are balanced (Fig.…”

“…OsHKT2;4 Defines a New Functional Type of Transporter within the HKT Family HKT transporters have been sorted out into two subfamilies based on phylogenetic analyses and functional differences observed in heterologous systems : Subfamily 1 HKT transporters are thought to be Na + selective whereas subfamily 2 transporters are expected to transport both Na + and K + (Rubio et al, 1995;Uozumi et al, 2000;Haro et al, 2005;Ren et al, 2005;Jabnoune et al, 2009). Apart from OsHKT2;4, all the members of subfamily 2 transporters that have been characterized so far are close homologs of TaHKT2;1 Hauser and Horie, 2010), the first subfamily 2 transporter characterized (Rubio et al, 1995).…”

Sodium as nutrient and toxicant

“…However, only very limited (and misleading; see Haro et al 2005) demonstrations of such a function outside heterologous expression systems, such as Xenopus oocytes and yeast cells, have thus far occurred (Rubio et al 1995;Spalding et al 1999;Haro et al 2005). Other than these instances, little evidence for Na + -coupled K + uptake exists in terrestrial plants Rodríguez-Navarro and Rubio 2006;Corratgé-Faillie et al 2010;Schulze et al 2012), although it may play a significant role in aquatic angiosperms and algae .…”

“…Na + -coupled transport systems are very common in animals and marine protists, and one wonders how much flexibility might exist in other transport systems that are normally coupled to H + gradients (Britto and Kronzucker 2005), to utilize Na + gradients under special circumstances. This possibility is suggested, for instance, by the demonstration, in heterologous systems, such as Xenopus oocytes (albeit not necessarily in planta; Walker et al 1996;Box and Schachtman 2000), that K + transporters of the HKT family, normally acting as uniporters or H + -coupled mechanisms, can undergo Na + coupling (Rubio et al 1995;Haro et al 2005). Another intriguing suggestion is that fluxes of both phosphate and nitrate might be coupled to the steep electrochemical potential gradient for Na + in the marine angiosperm Zostera marinus .…”