Potassium and sodium transport in non-animal cells: the Trk/Ktr/HKT transporter family.

Bacterial Trk and Ktr, fungal Trk and plant HKT form a family of membrane transporters permeable to K(+) and/or Na(+) and characterized by a common structure probably derived from an ancestral K(+) channel subunit. This transporter family, specific of non-animal cells, displays a large diversity in terms of ionic permeability, affinity and energetic coupling (H(+)-K(+) or Na(+)-K(+) symport, K(+) or Na(+) uniport), which might reflect a high need for adaptation in organisms living in fluctuating or dilute environments. Trk/Ktr/HKT transporters are involved in diverse functions, from K(+) or Na(+) uptake to membrane potential control, adaptation to osmotic or salt stress, or Na(+) recirculation from shoots to roots in plants. Structural analyses of bacterial Ktr point to multimeric structures physically interacting with regulatory subunits. Elucidation of Trk/Ktr/HKT protein structures along with characterization of mutated transporters could highlight functional and evolutionary relationships between ion channels and transporters displaying channel-like features.

A procedure for localisation and electrophysiological characterisation of ion channels heterologously expressed in a plant context.

BACKGROUND: In silico analyses based on sequence similarities with animal channels have identified a large number of plant genes likely to encode ion channels. The attempts made to characterise such putative plant channels at the functional level have most often relied on electrophysiological analyses in classical expression systems, such as Xenopus oocytes or mammalian cells. In a number of cases, these expression systems have failed so far to provide functional data and one can speculate that using a plant expression system instead of an animal one might provide a more efficient way towards functional characterisation of plant channels, and a more realistic context to investigate regulation of plant channels. RESULTS: With the aim of developing a plant expression system readily amenable to electrophysiological analyses, we optimised experimental conditions for preparation and transformation of tobacco mesophyll protoplasts and engineered expression plasmids, that were designed to allow subcellular localisation and functional characterisation of ion channels eventually in presence of their putative (possibly over-expressed) regulatory partners. Two inward K+ channels from the Shaker family were functionally expressed in this system: not only the compliant KAT1 but also the recalcitrant AKT1 channel, which remains electrically silent when expressed in Xenopus oocytes or in mammalian cells. CONCLUSION: The level of endogenous currents in control protoplasts seems compatible with the use of the described experimental procedures for the characterisation of plant ion channels, by studying for instance their subcellular localisation, functional properties, structure-function relationships, interacting partners and regulation, very likely in a more realistic context than the classically used animal systems.

Hyperpolarization-activated calcium channels at the tip of Arabidopsis root hairs.

The root hair elongative growth phase ("tip growth"), like that of other tip-growing systems such as pollen tubes, algal rhizoids, and fungal hyphae, is associated with an apex-high cytosolic free calcium ([Ca(2+)](c)) gradient generated by a local Ca(2+) influx at the tip. This gradient has been shown to be a fundamental regulator of tip growth. Here, we have performed patch-clamp experiments at root hair apices of Arabidopsis thaliana (after localized cell wall laser ablation) to characterize the plasma membrane Ca(2+) channels implicated in the tip Ca(2+) influx. We have identified a hyperpolarization-activated Ca(2+) conductance. This conductance is selective for Ca(2+) over K(+) and Cl(-) (P(Ca)/P(K) = 15; P(Ca)/P(Cl) = 25) and is fully blocked by < 100-microM trivalent cations (La(3+), Al(3+), Gd(3+)). The selectivity sequence among divalent cations (determined by comparisons of the channel unitary conductance) is Ba(2+) > Ca(2+) (22 pS in 10 mM) approximately Mg(2+) > Mn(2+). This conductance was operative at typical growing hair apical resting membrane potentials. Moreover, it was seen to be down-regulated in growing hair subapical regions, as well as at the tip of mature hairs (known not to exhibit Ca(2+) influx). We therefore propose that this inward-rectifying Ca(2+) conductance is inherently involved in the apical Ca(2+) influx of growing hairs. The observed enhancement of the conductance by increased [Ca(2+)](c) may form part of a positive feedback system for continued apical Ca(2+) influx during tip growth.

Magnesium ions promote assembly of channel-like structures from beticolin 0, a non-peptide fungal toxin purified from Cercospora beticola.

Beticolins are toxins produced by the fungus Cercospora beticola. Using beticolin 0 (B0), we have produced a strong and Mg(2+)-dependent increase in the membrane conductance of Arabidopsis protoplasts and Xenopus oocytes. In protein-free artificial bilayers, discrete deflexions of current were observed (12 pS unitary conductance in symmetrical 100 mM KCl) in the presence of B0 (approximately 10 microM) and in the presence of nominal Mg2+. Addition of 50 microM Mg2+ induced a macroscopic current which could be reversed to single channel current by chelating Mg2+ with EDTA. Both unitary and macroscopic currents were ohmic. The increase in conductance of biological membranes triggered by B0 is therefore likely to originate from the ability of this toxin to organize itself into transmembrane pores in the presence of Mg2+. The pore is poorly selective, displaying permeability ratios PCl/PK, PNa/PK and PCa/PK close to 0.3, 0.65 and 0.4, respectively. Such channel-like activity could be involved in the deleterious biological activity of the toxin, by causing the collapse of ionic and electrical gradients through biological membranes together with Ca2+ influx and scrambling of cellular signals.

Laser microsurgery permits fungal plasma membrane single-ion-channel resolution at the hyphal tip.

A method for formation of high-electrical-resistance seals on the Neurospora crassa plasma membrane, allowing resolution of single-ion-channel activity by patch clamp electrophysiology, is reported. Laser microsurgery permits access to the hyphal apex without enzymatic cell wall digestion and loss of morphological polarity. Cell wall reformation is delayed by brefeldin. This method can allow full characterization of apical plasma membrane channels, which are implicated in tip growth.

Expression of a cloned plant K+ channel in Xenopus oocytes: analysis of macroscopic currents.

The open reading frame from the Arabidopsis thaliana KAT1 cDNA was cloned in a transcription plasmid between the 3' and 5' untranslated regions of a beta-globin cDNA from Xenopus oocyte. The polyadenylated transcripts resulting from in vitro transcription gave rise to high levels of expression of KAT1 channel when injected in Xenopus oocytes. Upon hyperpolarization, a slow activating current could be recorded, inwardly- or outwardly-directed, depending on K+ external concentration. Predictions of the voltage-gated channel theory were shown to fit the data well. The equivalent gating charge and the half-activation potential ranged around 2 and -145 mV, respectively. KAT1 gating characteristics did not depend on K+ external concentration nor on external pH in the 5.0-7.5 range. KAT1 conductance was, however, increased (40%) when external pH was decreased from 6.5 to 5.0. The apparent affinity constant of KAT1 for K+ lay in the range 15-30 mM, at external pH 7.4. As for many K+ channels of animal cells, external caesium caused a voltage-dependent blockage of inward (but not outward) KAT1 current, whereas tetraethylammonium caused a voltage-independent block of both inward and outward KAT1 currents. In conclusion, high levels of expression made it possible to carry out the first quantitative analysis of KAT1 macroscopic currents. KAT1 channel was shown to display features similar to those of as yet uncloned inward-rectifying voltage-gated channels described in both plant cells (namely guard cells) and animal cells.

Level of expression in Xenopus oocytes affects some characteristics of a plant inward-rectifying voltage-gated K+ channel.

The plant K+ channel KAT1 shows some similarity to animal voltage-gated channels of the Shaker superfamily. Contrary to these animal counterparts, this plant channel is inwardly rectifying, being gated upon hyperpolarization. Different levels of expression of KAT1 in Xenopus oocytes could be obtained by increasing the amount of injected cRNA. The resulting KAT1 gating and sensitivity to external caesium were significantly changed. Similar findings have been published regarding animal voltage-gated channels. The present data show that plant channels may also undergo modification of their activity upon modification of their level of expression.

Coupling of water and potassium ions in K channels of the tonoplast of Chara.

Electrokinetic measurements, of streaming potential, were carried out on an excised inside-out patch of the vacuolar membrane of Chara corallina. A water activity gradient was imposed across the patch membrane containing a single K(+) channel by addition of sorbitol to one side. Two different K(+) channels were found in the tonoplast. Their open channel conductance was investigated as a function of KCl concentration. They had a maximal open channel conductance of 247 and 173 pS, and an apparent affinity (K(M)) of 116 and 92 mM, respectively. Single-channel zero-current potentials were determined in the presence of an osmotic gradient, and dilution artifacts were corrected for by addition of valinomycin to the bath. Our results suggest that 29 water molecules were coupled to the transport of one K(+) ion in the large conductance K(+) channel which has a pore radius of approximately 1.5 nm.