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Journal of Plant Physiology 171 (2014) 723–731
Contents lists available at ScienceDirect
Journal of Plant Physiology
journal homepage: www.elsevier.com/locate/jplph
Physiology
The twins K+ and Na+ in plants夽
˜ Benito a,1 , Rosario Haro a,1 , Anna Amtmann b , Tracey Ann Cuin c , Ingo Dreyer a,∗
Begona
a
Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Madrid, Spain
Institute of Molecular, Cellular and Systems Biology (MCSB), College of Medical, Veterinary and Life Sciences (MVLS), University of Glasgow, Glasgow, UK
c
Biochimie et Physiologie Moléculaire des Plantes, Institut de Biologie Intégrative des Plantes, UMR 5004 CNRS/UMR 0386 INRA/Montpellier
SupAgro/Université Montpellier 2, Montpellier, France
b
a r t i c l e
i n f o
Article history:
Received 26 July 2013
Received in revised form 1 October 2013
Accepted 2 October 2013
Available online 3 March 2014
Keywords:
HKT
Potassium
Sodium
Transport mechanism
a b s t r a c t
In the earth’s crust and in seawater, K+ and Na+ are by far the most available monovalent inorganic
cations. Physico-chemically, K+ and Na+ are very similar, but K+ is widely used by plants whereas Na+
can easily reach toxic levels. Indeed, salinity is one of the major and growing threats to agricultural
production. In this article, we outline the fundamental bases for the differences between Na+ and K+ .
We present the foundation of transporter selectivity and summarize ﬁndings on transporters of the HKT
type, which are reported to transport Na+ and/or Na+ and K+ , and may play a central role in Na+ utilization
and detoxiﬁcation in plants. Based on the structural differences in the hydration shells of K+ and Na+ , and
by comparison with sodium channels, we present an ad hoc mechanistic model that can account for ion
permeation through HKTs.
© 2013 Elsevier GmbH. All rights reserved.
Introduction
Potassium with ∼2.1% (position 8) and sodium with ∼2.4% (position 6) are among the top ten elements present in the continental
crust (position 1: oxygen 47.2%, 2: silicon 28.8%, 3: aluminum
8.0%, 4: iron 4.3%, 5: calcium 3.9%, 7: magnesium 2.2%, 9: titanium 0.4%, and 10: carbon 0.2%; Wedepohl, 1995). They are both far
more abundant than the other alkali metals rubidium, lithium, and
cesium (all <0.01%). Also in seawater, the monovalent cations Na+
and K+ are amongst the major dissolved ions. There, however, Na+
dominates together with Cl− (both ∼500 mM) over Mg2+ (∼50 mM),
SO4 2− (∼28 mM), Ca2+ (∼10 mM) and K+ (∼10 mM; values are for
average seawater of salinity 352 ; Riley and Tongudai, 1967). Under
these conditions, primitive cells might have found that accumulation of K+ along with exclusion of the more abundant Na+ provided
Abbreviations: HAK, high-afﬁnity K+ uptake transporter (name of a transporter
family); HKT, high-afﬁnity K+ transporter (name of a transporter family); KT, K+
transporter (name of a transporter family); Ktr, K+ transporter (name of a transporter
family); Kup, K+ uptake transporter (name of a transporter family); P, pore-forming
region; TM, transmembrane ␣-helix; Trk, transport of K+ (name of a transporter
family).
夽 This article is part of a Special Issue entitled “Potassium effect in plants”.
∗ Corresponding author. Tel.: +34 913364588.
E-mail address: [email protected] (I. Dreyer).
1
These authors contributed equally to this work and are listed alphabetically.
2
The Practical Salinity Scale deﬁnes salinity in terms of the conductivity ratio of
a sample to that of a solution of 32.4356 g of KCl at 15 ◦ C in a 1 kg solution. A sample
of seawater at 15 ◦ C with conductivity equal to this KCl solution has a salinity of
exactly 35 practical salinity units (psu).
0176-1617/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.jplph.2013.10.014
an efﬁcient way to energize the plasma membrane. That the inorganic composition of the cytoplasm appears to be highly conserved
for all eukaryotic organisms indicates that already at the outset of
evolution in seawater, living cells developed a preference among
the inorganic monovalent cations for K+ over Na+ for essential
functions such as, for example, maintaining electro-neutrality and
osmotic equilibrium. K+ therefore became an indispensable necessity for living cells; a dependency also inherited by Embryophyta,
which need to survive in oligotrophic environments where K+ is
present at much lower concentrations than in seawater (see Zörb
et al., 2014, for more details on the bioavailability of K+ in soils).
This feature remained also conserved in higher plant species that
returned to the sea after their predecessors spent a period of time
on land as the sea grass Posidonia oceanica, for instance (Carpaneto
et al., 1997, 1999, 2004).
In plants, potassium plays a vital role in a wide range of both
biophysical and biochemical processes. It exists as a monovalent
cation and does not participate in covalent binding; it functions
to maintain charge balance. The preservation of cell turgor pressure is very sensitive to a limited K+ supply. Indeed, due to its
high mobility, K+ is usually the principle cation that contributes
to vacuole and cell expansion (for K+ transporters in vacuoles see
Hamamoto and Uozumi, 2014; Pottosin and Dobrovinskaya, 2014).
Nonetheless, over a longer time scale it can be replaced by Na+
(Jeschke and Wolf, 1988) and/or organic solutes (Talbott and Zeiger,
1996), explaining the observed highly variable (10–200 mM) vacuolar K+ levels. In contrast, cytoplasmic levels are relatively stable,
near 100 mM (Leigh and Wyn Jones, 1984). It is suggested that as
total tissue K+ concentration declines, the cytoplasm maintains a
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B. Benito et al. / Journal of Plant Physiology 171 (2014) 723–731
homeostatic concentration of K+ to enable K-dependent processes
(Leigh and Wyn Jones, 1986; Walker et al., 1996; see also Anschütz
et al., 2014). Consequently, any initial changes in tissue K+ concentrations are likely to be at the expense of vacuolar K+ , with
other solutes being diverted to the vacuole to maintain the osmotic
potential.
At the biochemical level, potassium ions play an important role
in the activation of many enzymes, especially in protein and starch
synthesis, as well as in respiratory and photosynthetic metabolism
(Läuchli and Pﬂüger, 1979; Wyn Jones et al., 1979; Marschner,
2010). Starch synthesis, for instance, has a requirement of about
50 mM K+ for normal activity. In line with this level, most Krequiring enzymes need about 10–50 mM of K+ for optimal activity.
Interestingly, these levels may be reduced by the substitution of
other monovalent cations for K+ (Evans and Sorger, 1966; Wyn
Jones and Pollard, 1983). For example, Rb+ , Cs+ , and NH4 + are about
80% as effective as K+ at maintaining starch synthetase activity,
while Na+ is approximately 20% as effective (Nitsos and Evans,
1969). Hence, in both biophysical and biochemical processes, K+
may be the most efﬁcient monovalent cation, although to a certain
extent, it can be replaced.
Many halophytic plants are able to take advantage of the close
similarity between Na+ and K+ , and have adapted to grow in areas
of high salt. The presence of Na+ in the environment and its uptake
by plants can reduce the amount of K+ required to meet the plant’s
basic metabolic requirements. In addition, many non-halophytic
plants are able to metabolically utilize Na+ to some degree. Under a
limited K+ supply for example, Na+ (together with Mg2+ and Ca2+ )
can replace K+ in the vacuole as an alternative inorganic osmoticum
(Flowers and Läuchli, 1983; Kronzucker and Britto, 2011). The
released K+ is then available for more K-speciﬁc processes. That
the presence of tissue Na+ is associated with a reduced K+ content
in the leaves illustrates that Na+ can replace K+ in many functions,
so reducing the total amount of K+ required by the plant. Sodium
can alleviate K-deﬁciency symptoms and may decrease the critical foliar K+ concentration at which K-deﬁciency symptoms appear
(Besford, 1978). The application of Na+ to the growth medium has
been shown to stimulate the growth of many species (e.g. red beet;
Subbarao et al., 2003), and for some crops, e.g. celery, carrots and
forage crops, Na+ has been reported to even improve the quality
of the product. This indicates that some of the metabolic effects of
Na+ can be of practical value (Marschner, 2010).
Nevertheless, although many plants appear to utilize Na+ to a
certain degree, its excessive abundance is problematic (Kronzucker
et al., 2013). Under saline conditions, the growth of many plants
is limited due to an inadequate water availability and speciﬁc ion
toxicity at high Na+ levels; the plant cell cytosol rarely tolerates
Na+ concentrations above 20 mM (Walker et al., 1996; Amtmann
and Sanders, 1999; Blumwald et al., 2000). In many biochemical processes, Na+ is not as efﬁcient as K+ : while interference
with normal metabolic functioning may be tolerated to a certain
degree, Na+ does reach a noxious state at higher levels. Even natrophilic species such as Atriplex and Salicornia, cannot tolerate high
levels of cytoplasmic Na+ (Greenway and Osmond, 1972). Consequently, although the full role of Na+ in plant metabolism still
awaits full resolution, the general assumption related to Na+ tolerance among plants is that they compartmentalize and safely lock
the absorbed Na+ in vacuoles, using it as an inorganic osmoticum
in place of, or along with K+ (Bonales-Alatorre et al., 2013b).
Na+ uptake under saline conditions therefore mostly reﬂects the
need for an osmoticum during osmotic adjustment to salinity
stress. When Na+ is present in excess, i.e. under long lasting salt
stress conditions, the compartmentalization capacity will eventually reach its limit. To maintain an appropriately high K+ /Na+
ratio in leaves, Na+ needs to be excluded from photosynthetically active tissues and can be transported via the phloem from
shoot tissues to roots (Berthomieu et al., 2003; Hauser and Horie,
2010).
In this article, we outline the fundamental bases for the differences between the two monovalent alkali cations, Na+ and K+ . We
present the foundation of transporter selectivity and summarize
ﬁndings on transporters of the HKT-type that are reported to transport Na+ and/or Na+ and K+ (Table 1) and may play a central role in
Na+ utilization and detoxiﬁcation in plants. Based on the structural
differences of the hydration shells of K+ and Na+ , and by comparison with sodium channels from bacteria we propose a model for
ion permeation through HKTs.
Physicochemical resemblance of Na+ and K+
Sodium and potassium are structurally and chemically very similar elements and exhibit homologous behavior. Both are alkali
metals and belong to the s-block in the periodic table (just like
the earth alkali metals calcium and magnesium). This means that
their valence electrons are occupying s-orbitals, correlating with a
low ﬁrst ionization energy. Thus, sodium and potassium are never
found in their elemental forms in nature, but readily lose their outermost electron to form monovalent Na+ and K+ cations, and reach
a noble gas conﬁguration. Due to the fully ﬁlled 1s/2s/2p (Na+ ) and
1s/2s/2p/3s/3p (K+ ) orbitals of the electron shell, their tendency to
participate in covalent binding is very low. According to quantum
mechanical principles, an atom or an ion does not possess a deﬁned
edge, so an absolute value for the ionic radii of Na+ and K+ cannot
be provided. However, based on estimates from crystal structures,
effective ionic radii can be derived (Shannon, 1976). Depending on
the method and on the coordination of the ions, the values provided for Na+ range from 1.02 A˚ to 1.16 A˚ and for K+ , from 1.38 A˚
˚ Thus, K+ is ∼1.3 times larger than Na+ . The similarities
to 1.52 A.
between Na+ and K+ may explain their interchangeability in some
physiological processes, but the small physicochemical differences
may not explain their distinct roles in others.
For biological processes, it is very important to take into account
the interaction of ions with polar water molecules. Indeed, when
estimating the radii of the ions in water, it was shown that the
proportions change. The radius of the hydrated Na+ was estimated
to be 358 pm, while the K+ ion has a radius of 331 pm (Volkov
et al., 1997). Additionally, ion hydration plays an important role in
chemical processes such as electrostriction. Therefore, particular
differences between Na+ and K+ may depend on the details of their
hydration shells. From theoretical analyses of the energetic contributions of each hydration shell to the total enthalpy of hydration
(−322 kJ/mol for K+ and −406 kJ/mol for Na+ ), it is proposed that
the hydrated K+ and Na+ cations have a distinct behavior (CarrilloTripp et al., 2003). In solution, Na+ ions have two clearly deﬁned
hydration shells; the hydration number of the ﬁrst shell is 5–6, and
that of the second ∼16. In contrast, a potential second hydration
shell of K+ is very loosely marked; the hydration number of the
ﬁrst shell is 7–8 (Fig. 1). Thus, the most striking energy difference
between K+ and Na+ resides in the ﬁrst shell. In the case of sodium,
water molecules have a very strong interaction with the ion and
a weak interaction with the rest of the solvent. On the contrary,
in potassium, the interaction with the ion is almost the same as
the interaction with the solvent. This weaker interaction of K+ with
its ﬁrst hydration shell equips it with a larger structural ﬂexibility (Carrillo-Tripp et al., 2003; Mancinelli et al., 2007). K+ allows
for a greater distortion of its hydration shell, which enables easier
accommodation to different hydration or hydration-replacement
cages (Carrillo-Tripp et al., 2006). Na+ in contrast, requires a cage
very similar to its own surroundings in the solvent. In interactions with ﬂexible partners such as proteins, Na+ tends to distort
its surroundings more than K+ does. This might explain why Na+
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Table 1
Transport characteristics of HKTs from different species.
Species
Name
Transported ion
Functional characterization in
Oocytes
Yeast
Other systems
AtHKT1;1
Na+ , low afﬁnity K+ uptake in E. coli
Yes
Yes
E. coli, knockout plants
Eucalyptus
camaldulensis
Hordeum vulgare
Mesembryanthemum
crystallinum
Oryza sativa
EcHKT1;1
EcHKT1;2
HvHKT2;1
McHKT1;1
K+ , Na+
K+ , Na+
Na+ , K+
K+ , Na+
Yes
Yes
No
Yes
No
No
Yes
Growth testsa
Growth tests in E. coli
Growth tests in E. coli
OsHKT2;1
Na+ , K+ currents in oocytes
Yes
Yes
Tobacco cells, knockout plants
OsHKT1;5
OsHKT2;4
OsHKT1;1
OsHKT1;3
OsHKT2;2
Na+
Predominantly K+ , Na+
Low afﬁnity Na+ , competitively inhibited by K+
Na+
K+ , Na+
Yes
Yesb
Yes
Yes
Yes
No
Growth testsa
Yes
No
No
Tobacco cells
Os-HKT2;2/1
K+ , Na+
Yes
Yes
PhaHKT1
PpHKT1
PutHKT2;1
SlHKT1;2
TsHKT1;2
TaHKT2;1
K+ , Na+
Na+
K+ , Na+
Na+
K+ , Na+
K+ , Na+
No
No
No
No
No
Yes
Growth testsa
Yes
Yes
Yes
Growth testsa
Yes
O. sativa
Pokkali
O. sativa
Pokkali var. Nonabokra
Phragmites australis
Physcomitrella patens
Pucinellia tenuiﬂora
Solanum lycopersicum
Thellungiella salsuginea
Triticum aestivum
Uozumi et al. (2000), Rus et al. (2001), Mäser et al. (2002a),
Berthomieu et al. (2003), and Xue et al. (2011)
Fairbairn et al. (2000) and Liu et al. (2001)
Fairbairn et al. (2000) and Liu et al. (2001)
Haro et al. (2005) and Banuelos et al. (2008)
Su et al. (2003)
Horie et al. (2001), Golldack et al. (2002), Garciadeblas et al.
(2003), Horie et al. (2007), Jabnoune et al. (2009), and Yao et al.
(2010)
Ren et al. (2005)
Lan et al. (2010), Horie et al. (2011), and Sassi et al. (2012)
Garciadeblas et al. (2003) and Jabnoune et al. (2009)
Jabnoune et al. (2009)
Horie et al. (2001) and Yao et al. (2010)
Oomen et al. (2012)
Knockout plants
Overexpression in plants
Overexpression in plants
Takahashi et al. (2007)
Haro et al. (2010)
Ardie et al. (2009)
Asins et al. (2013)
Ali et al. (2012)
Schachtman and Schroeder (1994), Rubio et al. (1995), and
Gassman et al. (1996)
B. Benito et al. / Journal of Plant Physiology 171 (2014) 723–731
Arabidopsis thaliana
References
Complementation of a K+ uptake-deﬁcient yeast strain was monitored; no further ion transport analysis has been carried out.
Two studies (Lan et al., 2010; Horie et al., 2011) also assigned a Ca2+ and Mg2+ permeability to OsHKT2;4 when expressed in oocytes. However, this could not be conﬁrmed in a careful reinvestigation (Sassi et al., 2012).
Instead, it is proposed that the Ca2+ /Mg2+ permeability results from the activation of an endogenous oocyte conductance.
a
b
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Fig. 1. Structures of the hydration shells of K+ and Na+ in free solution. Computational simulations (Carrillo-Tripp et al., 2003) indicate that, in solution, Na+ ions
have two clearly deﬁned hydration shells. The hydration number of the ﬁrst shell is
5–6, and that of the second ∼16. The hydration number of the ﬁrst shell of K+ is 7–8.
A potential second hydration shell of K+ is very loosely marked.
cannot or can only partially replace K+ as a coordinating ion in certain enzymatic reactions, whereas Rb+ or NH4 + , which have features
much more similar to K+ , are far more efﬁcient.
Structural basis for selective transmembrane K+ transport
Physiological data indicate that plants have a higher capacity for K+ uptake and distribution than for Na+ (for details on K+
uptake and distribution in plants see Nieves-Cordones et al., 2014;
Ahmad and Maathuis, 2014; Wigoda et al., 2014). Potassium ions
can be efﬁciently separated from Na+ , as demonstrated for potassium channels. Organisms from all kingdoms have such transporter
proteins, which under physiological conditions, are extraordinarily selective for potassium over other ions. The crystal structures
obtained for bacterial and mammalian K+ channels provide very
detailed insights into the selectivity mechanism (Doyle et al., 1998;
Kuo et al., 2003; Jiang et al., 2003; Long et al., 2005). The central permeation pathway of a channel is formed by a 4-fold TM1-P-TM2
motif of two transmembrane ␣-helices ﬂanking the pore helix (a
short ␣-helical stretch) and a highly conserved sequence, TxGYGD
(Fig. 2). The backbone carbonyl oxygen atoms of these ﬁve residues
face the center of the narrowest part of the permeation pathway,
while the side chains project away from the pore axis. The P-regions
of all four TM1-P-TM2 motifs contribute with their electronegative
carbonyls to form ﬁve similar, almost equidistantly spaced rings,
which provide four binding sites for K+ ions in between them. The
remainder of the channel is less selective and its wall is formed
by side chains of the residues of all four TM2s. The narrow selectivity ﬁlter closely matches the geometry, charge densities and atomic
distances that are typical of the inner hydration shell around the K+
ions when in free solution. During permeation, the oxygen atoms
of the channel backbone can substitute for water molecules and
coordinate the K+ ion (Fig. 2C). This allows K+ ions, but only K+
ions, to diffuse through the bottleneck of the channel as though in
free solution. Each K+ ion ‘hops’ from one site to the next, driven
by the repulsive electrical forces between ions in the pore, ﬁnally
regaining its hydration shell on leaving the oxygen cages. The selectivity ﬁlter effectively discriminates against ions with even minor
differences in charge density and ionic radius, including the smaller
Na+ ion. This is achieved without the need for a deep energy well
associated with binding, which would slow the rate of permeation.
The structure of the selectivity ﬁlter of potassium channels is
susceptible to disturbance. In mutagenesis studies, even conserved
exchanges modify the preference of plant K+ channels for K+ over
other alkali ions (Uozumi et al., 1995; Becker et al., 1996; Nakamura
et al., 1997; Dreyer et al., 1998; Ros et al., 1999; Moroni et al.,
Fig. 2. The pore of K+ channels. (A) A structural TM1-P-TM2 motif is built of
two transmembrane ␣-helices ﬂanking a short ␣-helical stretch, called the pore
helix, and the highly conserved sequence TxGYGD (schematic side view). (B) The
tetrameric assembly of four TM1-P-TM2 motifs results in the formation of a central
permeation pathway for K+ . The four TxGYGD stretches frame the narrowest part
of the pore (schematic top view). (C) The internal half of the permeation pathway
is lined by the C-terminal ends of the four TM2s. The hydration shells of the K+ ions
(green) can be replaced for the cages formed by the polar oxygen atoms (red) of
the backbone of the TxGYGD-stretches (side view; only two of the four subunits are
displayed).
2000; Nakamura and Gaber, 2009). The results indicate that not
only the pore-lining TxGYGD stretches but the entire P-region and
the neighboring parts of the ﬂanking TM2 also contribute to the ion
selectivity.
Another aspect of the selectivity ﬁlter is that it is not rigid; its
structure results from the interaction of the permeating ions with
the protein. It can dilate (Hoshi and Armstrong, 2013), and when
the K+ concentration drops, the permeation pathway becomes
unstable and collapses (Zhou et al., 2001). Such a collapse can
have important physiological consequences. In plants, several K+
channels make use of this phenomenon to adjust their activity to
the surrounding K+ concentration. Outward-rectifying K+ release
channels close when the driving force for potassium would favor
uptake (Johansson et al., 2006), and for inward-rectifying K+ uptake
channels, the collapsing pore acts a “valve” that blocks unwanted
K+ release. The threshold concentration, below which this block
happens, appears to be a characteristic channel feature. The pore
of AKT1-AtKC1 heteromers for example, collapses at higher K+
concentrations than that of AKT1 homomers, thus rendering the
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B. Benito et al. / Journal of Plant Physiology 171 (2014) 723–731
heteromers more efﬁcient in blocking K+ passage in the unfavorable
outward direction (Geiger et al., 2009).
In the absence of potassium, certain potassium channels can
conduct sodium (Korn and Ikeda, 1995), a Na+ ﬂux that is blocked
by the addition of low concentrations of K+ . The potassium afﬁnity, hence the ability to block the sodium current, varies among the
potassium channel subtypes and can be modulated by mutations in
the central permeation pathway (Starkus et al., 1997, 1998). Analysis of the permeation mechanism indicates that both sodium and
potassium can occupy the pore simultaneously and that multiple
occupancy results in interactions between ions in the channel pore
(Ogielska and Aldrich, 1998).
In this context, a recent report on a K+ channel cloned from a
salt-tolerant melon (Cucumis melo) cultivar provides new insights
into the issue of K+ /Na+ transport in plants. This channel appears
to be extraordinarily sensitive to Na+ (Zhang et al., 2011), whereas
related channels from model plants (e.g. Arabidopsis thaliana) are
almost inert towards Na+ (Dreyer and Uozumi, 2011). The “blocking
phenomenon” observed for the melon channel though, indicates
nothing more than Na+ entering the K+ transporter and potentially
permeating it. With this in mind, it can be speculated, despite the
lack of molecular data, that K+ channels partially permeable for Na+
may underlie the unaffected stomatal conductance in a cultivar of
Beta vulgaris, even when nearly 95% of the plant’s K+ is replaced by
Na+ and when Na+ levels in the leaves approach 200 mM (Subbarao
et al., 1999; for more details on the role of K+ in controlling stomatal
conductance see Blatt et al., 2014).
Nonetheless, during evolution, the selectivity of K+ over Na+ in
plant potassium channels appears not to be driven to its limits. A
study in which the selectivity ﬁlter of the Arabidopsis K+ channel
KAT1 was randomly mutagenized, identiﬁed mutations that confer an enhanced K+ /Na+ selectivity (Nakamura and Gaber, 2009).
It is so easy to improve K+ selectivity in laboratory experiments,
so it is likely that nature would have done this long ago, if it had
been beneﬁcial to the plant. It might not necessarily be an advantage to discriminate too strictly between K+ and Na+ . Indeed, there
are many transporters in plants that transport K+ along with other
cations, and some members of the family of Na+ /H+ antiporters
(NHXs) and cation/H+ antiporters (CHXs) also possess K+ transport activity (Yamaguchi et al., 2003; Chanroj et al., 2011; Lu et al.,
2011; Chanroj et al., 2012). In a similar manner, vacuolar and
plasma membrane non- or poorly selective cation channels (see
Demidchik, 2014; Pottosin and Dobrovinskaya, 2014) and transporters of the HKT-type (see below) discriminate less between K+
and Na+ or even select for Na+ over K+ . Thus, a plant’s strategy to
deal with the assets and drawbacks of the K+ /Na+ dualism might
be more to orchestrate the activity of these transporters (MuellerRoeber and Dreyer, 2007; Bonales-Alatorre et al., 2013a) rather than
improve their selectivity.
Members of the HKT family transport Na+ or both K+ and
Na+
As well as channels, bacteria, fungi and plants also possess
two speciﬁc families of K+ transporters: Trk/Ktr/HKT (Transport
of K+ [fungi, bacteria]; K+ transporter [bacteria]; High-afﬁnity K+
transporter [plants]) and HAK/Kup/KT (High-afﬁnity K+ uptake
[fungi, plants]; K+ uptake [bacteria]; K+ transporter [plants]), which
are both absent from animal cells. Details on the HAK-family are
provided elsewhere (Nieves-Cordones et al., 2014; Véry et al.,
2014). The HKT family comprises a large group of transporters
that have been identiﬁed in all plants studied up to now (Table 1;
Haro et al., 2010; see also Véry et al., 2014), even in non-ﬂowering
plants (Gomez-Porras et al., 2012). HKTs have been demonstrated
to contribute toward Na+ transport and could play a role in osmotic
727
adjustment and Na+ detoxiﬁcation. This group of monovalent
cation transporters was discovered in 1994 in wheat (Schachtman
and Schroeder, 1994), and its initial functional characterization in
yeast mutants defective in K+ uptake and in Xenopus laevis oocytes
suggested that it functions as a H+ –K+ symporter. Based on these
results, the family was called ‘High-afﬁnity K+ Transporter’, a designation that later turned out not to be fully appropriate. To date,
there is no experimental support that these transporters play a
signiﬁcant role in K+ transport in planta. Instead, HKTs isolated
from a wide variety of plant species are either Na+ uniporters or
Na+ –K+ symporters (Corratge-Faillie et al., 2010). They appear to
be key determinants for salt tolerance of plants rather than for K+
nutrition.
Nevertheless, the ﬁrst structural information on HKTs was
obtained by comparing them with the K+ channel KcsA from
Streptomyces lividans (Durell and Guy, 1999). Sequence analyses and hydrophobic transmembrane proﬁles support the notion
that HKT/TRK/Ktr proteins share a common structure of four
successively arranged protomeric TM1-P-TM2 motifs in a single
polypeptide chain, which strongly resembles the four assembled
identical subunits of KcsA. Similarly, HKTs form a central permeation pathway like K+ channels (Fig. 2B). This picture was further
substantiated by the crystal structure of a TrkH K+ transporter from
Vibrio parahaemolyticus, which is even more similar to HKTs (Cao
et al., 2011). Sequence comparison of known HKT proteins, together
with site-directed mutagenesis analyses, led to the proposal that
there is a conserved ‘selectivity ﬁlter’ motif. One subclass of HKTs
shows a Ser-Gly-Gly-Gly structure at the outer mouth of the narrowest part of the permeation pathway. In this structure, the ﬁrst
TM1-P-TM2 protomer contributes a serine residue, while the other
three have a glycine at that position. The other subclass is characterized by a Gly-Gly-Gly-Gly structure where all four protomers
contribute with a glycine. The Na+ permeability of HKTs was correlated with the Ser, but K+ permeability requires the presence of Gly
in the pore domain (Mäser et al., 2002b). However, there are exceptions to this rule such as the plant transporter OsHKT2;1 from rice,
EcHKT1;2 from Eucalyptus, McHKT1;1 from Mesembryantemum and
TsHKT1;2 from Thellungiella. All have been shown to be permeable
to K+ , despite their ﬁrst pore domain containing a Ser instead of a
Gly residue (Fairbairn et al., 2000; Su et al., 2003; Jabnoune et al.,
2009; Ali et al., 2012). The continuous and increasing number of
exceptions found indicates that K+ permeability in HKTs does not
depend only on the pore Gly residue; other structural elements
in addition to the key selectivity ﬁlter are likely to be involved in
determining the substrate selectivity of HKT proteins.
The permeability properties of HKTs are still far from being
understood (Corratge-Faillie et al., 2010; Cotsaftis et al., 2012). This
is mainly because the data are not as unequivocal as in the case of K+
channels. HKTs were functionally characterized after heterologous
expression in Xenopus oocytes and yeast, and were shown to mediate high-afﬁnity K+ uptake and high or low-afﬁnity Na+ uptake,
depending on the transporter and external Na+ and K+ concentrations (Table 1). Some transporters function as Na+ –K+ symporters
at low external Na+ and K+ concentrations, as demonstrated by Na+ stimulated K+ uptake and K+ -stimulated Na+ uptake (Rubio et al.,
1995; Haro et al., 2005; Jabnoune et al., 2009). At high external
Na+ concentrations, some of these transporters no longer transport
K+ , becoming Na+ uniporters (Gassman et al., 1996; Horie et al.,
2001; Jabnoune et al., 2009). Interestingly, OsHKT2;2/1 identiﬁed
from the salt tolerant rice cultivar Nona Bokra, shows a strong permeability for Na+ and K+ , even at high external Na+ concentrations.
This channel/transporter may therefore contribute to salt tolerance
in Nona Bokra by enabling root K+ uptake under saline conditions
(Oomen et al., 2012). The switch between uniport and symport
may also be dependent on the protein density in the membrane.
When HvHKT1 from barley was expressed at high levels in yeast,
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B. Benito et al. / Journal of Plant Physiology 171 (2014) 723–731
it mediates K+ or Na+ uniport. However, when protein expression
was signiﬁcantly decreased, the transporter acted in the K+ /Na+ symport mode (Banuelos et al., 2008).
Ambiguity of Na+ /K+ transport by HKTs – dynamic
interactions of the permeating ions with the pore?
In order to explain the variable permeation properties, two
different mechanistic models were proposed for HKTs and their
close relatives of the Ktr and Trk families from bacteria and fungi:
(i) carrier-mediated transport by an alternating-access model
(Gassman et al., 1996; Rubio et al., 1999; Haro and RodriguezNavarro, 2002) and (ii) a pore model very similar to that of K+
channels (Durell and Guy, 1999; Tholema et al., 2005; Corratge
et al., 2007). Although the alternating access model may account for
the different uniport and symport modes, it can hardly explain the
large currents measured after expressing HKTs in oocytes. Oomen
et al. (2012) describe currents for OsHKT2;1 from rice in the range
of 5–10 ␮A. This means that 30–60 × 1012 ions are transported per
second across the oocyte membrane. From this value, the transport
rate per protein can be estimated by taking into account the protein expression level. For comparison, a normal expression level
of plant K+ channels is 1–100 × 106 channels per oocyte. Assuming now for HKTs even a very high functional protein expression
level of up to 1010 transporters per cell, this would imply a transport rate of the carrier molecule of 3000–6000 ions per second; a
value that is 6–12 times higher than that of other plant carriers
(∼500 s−1 ; Carpaneto et al., 2010; Derrer et al., 2013). Considering
more realistic expression levels (e.g. ∼107 proteins per oocyte), the
turnover rate reaches values around 106 ions per second, which is
in the range of ion channels. Thus, HKTs very likely function as ion
channels.
As outline already before, the channel hypothesis is further
supported by structural homology models based on the crystal structure of the bacterial K+ channel KcsA (Durell and Guy,
1999) and by the crystal structure of a TrkH K+ transporter from
V. parahaemolyticus (Cao et al., 2011), which provided the basis
for homology models for OsHKT1;5 from Oryza sativa (rice) and
EcHKT1;2 from Eucalyptus camaldulensis (Waters et al., 2013).
Nonetheless, the variable dynamic permeation properties of HKTs
are hardly compatible with the relatively static selectivity mechanism of K+ channels.
To obtain insights into the reasons for the dynamic selectivity patterns of HKTs, it would be useful to sneak a peek at other
channel types. Potassium channels allow only the passage of fully
dehydrated K+ , while non-selective cation channels such as nicotinic acetylcholine receptors (nAChRs), transport hydrated ions
(Hille, 2001). Sodium channels appear to lie in between these two
extremes. In line with the special features of its hydration shell,
Na+ passes the vestibule of the selectivity ﬁlter in a partially dehydrated state. In particular, the ﬁrst hydration shell is not stripped
off (Qiu et al., 2012). Furthermore, in contrast to the four coordination sites found in K+ channels (Fig. 2), sodium channels appear to
have just two such sites and these differ in their ion coordination
properties (Zhang et al., 2013). During transit, a Na+ ion in the extracellular bulk solution moves close to the entrance of the pore (site
1) and when there, it interacts with a polarized side chain oxygen
atom in a pore residue, a serine for example. Thus, the permeating
Na+ ion only makes contact with one out of the four polypeptide
chains forming the selectivity ﬁlter. The ion then leaves site 1, slipping to site 2. There it is caged by eight carbonyl oxygen atoms
from the backbone of the pore-forming residues of all four protomers. The vacant site 1 can then be occupied by another Na+ ion
(Qiu et al., 2012; Zhang et al., 2013). Selectivity is achieved by the
energy barrier between the two coordination sites. A Ca2+ ion, for
instance, can be well coordinated at site 1 when entering from the
extracellular space, or at site 2 when coming from the intracellular side. A high-energy barrier, however, suppresses the transfer of
Ca2+ between the two sites, so Ca2+ blocks Na+ permeation (Zhang
et al., 2013). Interestingly, unlike K+ channels, the selectivity ﬁlter
of sodium channels appears to exhibit robust stability for various
ion conﬁgurations, even in the absence of ions (Qiu et al., 2012).
It is tempting to postulate for HKTs an analogous single ﬁle ion
permeation mechanism with two coordination sites for partially
dehydrated cations (Fig. 3). Such a ‘Na+ channel-based’ mechanism
would much better account for several experimental observations
than a ‘K+ channel-based’ model and could intuitively be supported
by the following considerations. The rigid energetic structure of the
ﬁrst hydration shell of Na+ hardly allows its stripping. Hence, it is
likely that Na+ permeates HKTs, as in sodium channels, only partly
dehydrated. The larger space required for a hydrated ion may imply
that the selectivity ﬁlter can accommodate only two ion coordination sites. These sites can be entered by hydrated Na+ and, due
to its ﬂexible hydration shell (Carrillo-Tripp et al., 2006), also by
K+ . The transition between the two coordination sites is hindered
by an internal barrier that represents an essential entity (but very
likely not the only one) of the selectivity ﬁlter. The detailed SerGly-Gly-Gly or Gly-Gly-Gly-Gly structure (Mäser et al., 2002b; also
see above) may constitute such a barrier. A serine residue with the
polar oxygen at its side chain may provide a preference for cation
coordination, whereas the glycine residues only provide space. The
afﬁnities of ions to the two coordination sites, together with the
energy barrier between them, would determine the permeability
of the pore. These parameters may depend on the one hand, on their
precise geometries being channel speciﬁc, but on the other hand,
the interaction of permeating ions with the coordination sites and
the barrier-forming side chains may slightly modify the topology.
These may be the reason for the observed concentration-dependent
differences in the apparent permeability of HKTs to Na+ and K+ .
The proposed model would account for the different transport
modes. The transporter may enable Na+ uniport (Fig. 3C, yellow
path) when only Na+ ions permeate along their electrochemical
gradients. Depending on the external Na+ concentration, this may
proceed either by the [0|Na] ↔ [0|0] ↔ [Na|0] ↔ [0|Na] cycle or via
the alternative [0|Na] ↔ [Na|Na] ↔ [Na|0] ↔ [0|Na] cycle. Additionally, the transporter may allow Na+ /K+ symport (Fig. 3C, green path)
via a [0|Na] ↔ [K|Na] ↔ [K|0] ↔ [0|K] ↔ [Na|K] ↔ [Na|0] ↔ [0|Na]
cycle. Finally, the transporter may theoretically also act as
a K+ uniporter (Fig. 3C, purple path) through the alternative [0|K] ↔ [0|0] ↔ [K|0] ↔ [0|K] and [0|K] ↔ [K|K] ↔ [K|0] ↔ [0|K]
cycles. The latter might be energetically unfavorable though due
to constraints in K+ ion coordination and/or barrier passage. The
model even explains the blocking effect of K+ on Na+ transport if
K+ ions pass the barrier [K|0] ↔ [0|K] much slower than Na+ . During
an experiment, the transporter is initially tested in the presence of
Na+ only, followed by the addition of K+ to the medium. Under the
ﬁrst, Na+ only condition, the relatively fast Na+ uniport is measured
(Fig. 3C, yellow path). Upon addition of K+ , some transporters not
only transport Na+ in uniport but also enter the mode of Na+ /K+
symport or K+ uniport, i.e. they are redirected from the yellow path
to the green or even purple one (Fig. 3C). This detour is accompanied by a reduced charge transport rate, so a smaller transport
current is measured.
The model is an ad hoc postulation. We should emphasize that
at the moment, the data basis for modeling permeation through
HKTs is very poor. The model may serve as guidance for future
structure-function studies on HKTs, aimed at clarifying their unique
permeation properties. Detailed insights into the mechanism of
Na+ (K+ ) transport could provide clues for comprehending the role
of HKTs in plant salt tolerance and probably also in Na+ and K+
nutrition.
Author's personal copy
B. Benito et al. / Journal of Plant Physiology 171 (2014) 723–731
729
Fig. 3. Structural differences in the hydration shell of K+ and Na+ point to different permeation mechanisms through ion channels. (A) Sketch of the pore-forming parts of
cation channels. Panels (B) and (C) focus on the narrowest part of the selectivity ﬁlter (dashed box). (B) Permeation of K+ through the selectivity ﬁlter of a K+ channel. When
entering the pore, the polar oxygen atoms of the backbone of the selectivity ﬁlter-forming stretch, TxGYGD, replace the hydration shell of K+ and take over the task of ion
coordination. Repulsive forces between the ions provoke their hopping through the four coordination sites. (C) Proposed model for ion permeation through the selectivity
ﬁlter of HKTs, inspired by the permeation process of Na+ ions through sodium channels. Na+ ions are coordinated by two coordination sites in a partially dehydrated form.
Due to energetic constraints, only the loosely bound second hydration shell is stripped off. Na+ thus retains the ﬁrst hydration shell. Nevertheless, one or two water molecules
of the shell might be substituted with polar oxygens at the coordination sites (e.g. the side chain of a serine residue; indicated in blue). The ﬂexible hydration shell of K+ also
allows the coordination of this ion in the two coordination sites. It is likely that coordination of Na+ or K+ at one site affects the ion coordination at the other site. This means
that the energetic conditions for the [0|0] → [K|0] transition is different from that of the [0|K] → [Na|K] and the [0|K] → [K|K] transitions, for example. Permeation can occur
as a Na+ uniport (yellow arrows; [Na|0] ↔ [0|Na] ↔ [Na|Na] ↔ [Na|0], when the external Na+ concentration is high; or [Na|0] ↔ [0|Na] ↔ [0|0] ↔ [Na|0], when the external Na+
concentration is low), as a Na+ /K+ symport (green arrows; [K|Na] ↔ [K|0] ↔ [0|K] ↔ [Na|K] ↔ [Na|0] ↔ [0|Na] ↔ [K|Na]), and theoretically as a K+ uniport (purple arrows; low
K+ levels: [K|0] ↔ [0|K] ↔ [0|0] ↔ [K|0]; high K+ levels: [K|0] ↔ [0|K] ↔ [K|K] ↔ [K|0]), albeit the latter might be energetically disfavored by hardly allowing the occupancy of
both coordination sites by K+ .
Conclusions and outlook
Na+ and K+ share many commonalities, but they also exhibit
clear differences. For plants, sodium is not per se, a toxic element
and here Paracelsus’ principle of toxicology ‘it’s the dose that makes
the poison’, holds true. Na+ can fulﬁll the biophysical function of
K+ as an osmoticum, provided the plants have the ability to take
up Na+ , translocate it to the shoot and sequester it in their vacuoles. In contrast to K+ transport, the mechanisms of Na+ transport
in plants are still poorly understood. Several types of transporter
are candidates for involvement in both Na+ and K+ transport. The
most evidence for a role in Na+ transport is available for HKTs, even
though they were initially characterized as high afﬁnity K+ transporters. Understanding the orchestration of all these transporters
is still a challenge to tackle. Nonetheless, understanding the assets
and drawbacks of the K+ /Na+ dualism may be a clue coping with
the agricultural problem of salinity and for developing strategies
allowing the reduction of K+ fertilizer by replacing it with cheaper,
in terms of money and energy, available Na+ .
Acknowledgements
This work was supported by grants from the Spanish Min˜
isterio de Economía y Competitividad to Ingo Dreyer, Begona
Benito and Rosario Haro (BFU2011-28815 and AGL2007-61705)
as well as a Marie Curie Career Integration Grant to Ingo Dreyer
(FP7-PEOPLE- 2011-CIG No. 303674–Regopoc), We are grateful to
Alonso Rodríguez-Navarro, Madrid, for helpful comments on the
manuscript.
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