K(+) channel profile and electrical properties of Arabidopsis root hairs.

Ion channels and solute transporters in the plasma membrane of root hairs are proposed to control nutrient uptake, osmoregulation and polar growth. Here we analyzed the molecular components of potassium transport in Arabidopsis root hairs by combining K(+)-selective electrodes, reverse transcription-PCR, and patch-clamp measurements. The two inward rectifiers AKT1 and ATKC1 as well as the outward rectifier GORK dominated the root hair K(+) channel pool. Root hairs of AKT1 and ATKC1 loss-of-function plants completely lack the K(+) uptake channel or exhibited altered properties, respectively. Upon oligochitin-elicitor treatment of root hairs, transient changes in K(+) fluxes and membrane polarization were recorded in wild-type plants, while akt1-1 root hairs showed a reduced amplitude and pronounced delay in the potassium re-uptake process. This indicates that AKT1 and ATKC1 represent essential alpha-subunits of the inward rectifier. Green fluorescent protein (GFP) fluorescence following ballistic bombardment with GORK promoter-GFP constructs as well as analysis of promoter-GUS lines identified this K(+) outward rectifier as a novel ion channel expressed in root hairs. Based on the expression profile and the electrical properties of the root hair plasma membrane we conclude that AKT1-, ATKC- and GORK-mediated potassium transport is essential for osmoregulation and repolarization of the membrane potential in response to elicitors.

KAT1 is not essential for stomatal opening.

It is generally accepted that K(+) uptake into guard cells via inward-rectifying K(+) channels is required for stomatal opening. To test whether the guard cell K(+) channel KAT1 is essential for stomatal opening, a knockout mutant, KAT1En-1, was isolated from an En-1 mutagenized Arabidopsis thaliana population. Stomatal action and K(+) uptake, however, were not impaired in KAT1-deficient plants. Reverse transcription-PCR experiments with isolated guard cell protoplasts showed that in addition to KAT1, the K(+) channels AKT1, AKT2/3, AtKC1, and KAT2 were expressed in this cell type. In impalement measurements, intact guard cells exhibited inward-rectifying K(+) currents across the plasma membrane of both wild-type and KAT1En-1 plants. This study demonstrates that multiple K(+) channel transcripts exist in guard cells and that KAT1 is not essential for stomatal action.

GORK, a delayed outward rectifier expressed in guard cells of Arabidopsis thaliana, is a K(+)-selective, K(+)-sensing ion channel.

Here we report on the molecular identification, guard cell expression and functional characterization of AtGORK, an Arabidopsis thaliana guard cell outward rectifying K(+) channel. GORK represents a new member of the plant Shaker K(+) channel superfamily. When heterologously expressed in Xenopus oocytes the gene product of GORK mediated depolarization-activated K(+) currents. In agreement with the delayed outward rectifier in intact guard cells and protoplasts thereof, GORK is activated in a voltage- and potassium-dependent manner. Furthermore, the single channel conductance and regulation of GORK in response to pH changes resembles the biophysical properties of the guard cell delayed outward rectifier. Thus GORK very likely represents the molecular entity for depolarization-induced potassium release from guard cells.

KDC1, a novel carrot root hair K+ channel. Cloning, characterization, and expression in mammalian cells.

Potassium is an essential nutrient which plays an important role in many aspects of plant growth and development. Plants have developed a number of highly specific mechanisms to take up potassium from the soil; these include the expression of K(+) transporters and potassium channels in root cells. Despite the fact that root epidermal and hair cells are in direct contact with the soil, the role of these tissues in K(+) uptake is not well understood. Here we report the molecular cloning and functional characterization of a novel potassium channel KDC1 which forms part of a new subfamily of plant K(in) channels. Kdc1 was isolated from carrot root RNA and in situ hybridization experiments show Kdc1 to be highly expressed in root hair cells. Expressing the KDC1 protein in Chinese hamster ovary cells identified it as a voltage and pH-dependent inwardly rectifying potassium channel. An electrophysiological analysis of carrot root hair protoplasts confirmed the biophysical properties of the Kdc1 gene product (KDC1) in the heterologous expression system. KDC1 thus represents a major K(+) uptake channel in carrot root hair cells.

Histidine(118) in the S2-S3 linker specifically controls activation of the KAT1 channel expressed in Xenopus oocytes.

The guard cell K(+) channel KAT1, cloned from Arabidopsis thaliana, is activated by hyperpolarization and regulated by a variety of physiological factors. Low internal pH accelerated the activation kinetics of the KAT1 channel expressed in Xenopus oocytes with a pK of approximately 6, similar to guard cells in vivo. Mutations of histidine-118 located in the putative cytoplasmic linker between the S2 and S3 segments profoundly affected the gating behavior and pH dependence. At pH 7.2, substitution with a negatively charged amino acid (glutamate, aspartate) specifically slowed the activation time course, whereas that with a positively charged amino acid (lysine, arginine) accelerated. These mutations did not alter the channel's deactivation time course or the gating behavior after the first opening. Introducing an uncharged amino acid (alanine, asparagine) at position 118 did not have any obvious effect on the activation kinetics at pH 7.2. The charged substitutions markedly decreased the sensitivity of the KAT1 channel to internal pH in the physiological range. We propose a linear kinetic scheme to account for the KAT1 activation time course at the voltages where the opening transitions dominate. Changes in one forward rate constant in the model adequately account for the effects of the mutations at position 118 in the S2-S3 linker segment. These results provide a molecular and biophysical basis for the diversity in the activation kinetics of inward rectifiers among different plant species.