Final Report Summary - REGOPOC (Regulation of Plant Potassium Channels)
The essential nutrient potassium (K+) is the mineral element with the highest concentration in plants, and together with nitrogen and phosphorous is limiting for plant production in many natural and agricultural habitats. The potassium concentration in normal soil (10µM-100µM) is about 3-4 orders of magnitude lower than in the plant. Therefore a plant has to invest energy for the uptake of K+ and its transport throughout the plant. Not surprisingly, plants possess a large number of genes encoding K+ transporters – including high-affinity transporters and ion channels – with diverse functional and regulatory properties. Distinct subsets of these transporters operate under different conditions and in different tissues. Significantly, it is estimated that K+ channels contribute to more than 50% of the nutritional K+ uptake under most field conditions.
Voltage-gated plant potassium channels belong to the superfamily of voltage-gated K+ channels. A functional K+ channel is a tetramer built up of four alpha-subunits. A single alpha-subunit shows a topology of six transmembrane domains (identified as S1–S6) flanked by cytosolic NH2- and COOH-termini, and a P (pore) loop and helix between S5 and S6 that contribute to the channel pore and ion selectivity filter. All channels belonging to the family of plant voltage-gated potassium channels share this structure. However, despite their common structure, at the functional (gating) level they are astonishingly diverse and segregate into four functionally different subfamilies: (i) Hyperpolarization-activated, inward-rectifying Kin channels open at negative membrane potentials and mediate potassium uptake energized by H+-ATPases that establish a very negative membrane voltage. (ii) Silent (Ksilent) channel subunits comprise a second group; they do not form functional channels when expressed on their own. However, they have a strong affinity for hetermerization with Kin subunits and affect the voltage-dependence of gating by displacing channel activation to more negative values. This negative shift in channel activation has been suggested as an adaptive mechanism under K+-limiting conditions; on its own, gating of the Kin homotetramer accommodates poorly for changes in external K+ concentration, and the more negative threshold of the heterotetramer avoids K+ loss through the channel assembly at intermediate voltages. (iii) Weak-rectifying Kweak channels display two current components with different voltage-dependent properties. One channel subpopulation showed gating analogous to that of K+-uptake channels opening at voltages more negative than around -100 mV (gating mode 1); the second showed little sensitivity to voltage, with channels being open over the entire physiological voltage range (gating mode 2). Despite some important differences, Kin, Kweak and Ksilent subunits are commonly associated with channel opening upon hyperpolarization to permit K+ influx. Thus, Kweak and Ksilent subunits can be considered as special cases within the broader grouping of inward-rectifying channels. The ability of Kin, Kweak and Ksilent subunits to assemble heteromeric channels with one another strongly favors this viewpoint. (iv) By contrast, Kout channels gate open (or activate) with membrane depolarization, thus giving rise to an outward rectification of the current–voltage curve for the channels. These outward-rectifying channels facilitate K+ efflux, usually in charge balance with anions passing through Cl- channels, and contribute to solute loading of the xylem and solute loss from guard cells during stomatal closure. Outward-rectifying K+ channels do not assemble with Kin, Kweak and Ksilent subunits. Furthermore, unlike the inward-rectifying assemblies, Kout channels exhibit a unique sensitivity of the gate to both membrane voltage and extracellular alkali cation concentration: the channels only open at voltages positive of EK (the Nernst-equilibrium voltage for K+), and so ensure K+ efflux regardless of the extracellular K+ concentration.
The REGOPOC project addressed aspects of the regulation of plant voltage-gated K+ channels at the protein level. In particular, the opposite rectification of Kin and Kout channels and the regulation of the gating mode of Kweak channels were investigated. Initial homology modeling and molecular dynamics simulations allowed the identification of domains in Kin and Kout channels that can be swapped without affected their functional properties. This strategy enabled the generation of a Kin-Kout channel pair that still exhibits its original function but that shares >90% identity at the protein level. This channel pair will allow fundamental insights into the gating process of potassium channels and may allow to fine-tune these channels types for better adaptation to particular environmental conditions.
Voltage-gated plant potassium channels belong to the superfamily of voltage-gated K+ channels. A functional K+ channel is a tetramer built up of four alpha-subunits. A single alpha-subunit shows a topology of six transmembrane domains (identified as S1–S6) flanked by cytosolic NH2- and COOH-termini, and a P (pore) loop and helix between S5 and S6 that contribute to the channel pore and ion selectivity filter. All channels belonging to the family of plant voltage-gated potassium channels share this structure. However, despite their common structure, at the functional (gating) level they are astonishingly diverse and segregate into four functionally different subfamilies: (i) Hyperpolarization-activated, inward-rectifying Kin channels open at negative membrane potentials and mediate potassium uptake energized by H+-ATPases that establish a very negative membrane voltage. (ii) Silent (Ksilent) channel subunits comprise a second group; they do not form functional channels when expressed on their own. However, they have a strong affinity for hetermerization with Kin subunits and affect the voltage-dependence of gating by displacing channel activation to more negative values. This negative shift in channel activation has been suggested as an adaptive mechanism under K+-limiting conditions; on its own, gating of the Kin homotetramer accommodates poorly for changes in external K+ concentration, and the more negative threshold of the heterotetramer avoids K+ loss through the channel assembly at intermediate voltages. (iii) Weak-rectifying Kweak channels display two current components with different voltage-dependent properties. One channel subpopulation showed gating analogous to that of K+-uptake channels opening at voltages more negative than around -100 mV (gating mode 1); the second showed little sensitivity to voltage, with channels being open over the entire physiological voltage range (gating mode 2). Despite some important differences, Kin, Kweak and Ksilent subunits are commonly associated with channel opening upon hyperpolarization to permit K+ influx. Thus, Kweak and Ksilent subunits can be considered as special cases within the broader grouping of inward-rectifying channels. The ability of Kin, Kweak and Ksilent subunits to assemble heteromeric channels with one another strongly favors this viewpoint. (iv) By contrast, Kout channels gate open (or activate) with membrane depolarization, thus giving rise to an outward rectification of the current–voltage curve for the channels. These outward-rectifying channels facilitate K+ efflux, usually in charge balance with anions passing through Cl- channels, and contribute to solute loading of the xylem and solute loss from guard cells during stomatal closure. Outward-rectifying K+ channels do not assemble with Kin, Kweak and Ksilent subunits. Furthermore, unlike the inward-rectifying assemblies, Kout channels exhibit a unique sensitivity of the gate to both membrane voltage and extracellular alkali cation concentration: the channels only open at voltages positive of EK (the Nernst-equilibrium voltage for K+), and so ensure K+ efflux regardless of the extracellular K+ concentration.
The REGOPOC project addressed aspects of the regulation of plant voltage-gated K+ channels at the protein level. In particular, the opposite rectification of Kin and Kout channels and the regulation of the gating mode of Kweak channels were investigated. Initial homology modeling and molecular dynamics simulations allowed the identification of domains in Kin and Kout channels that can be swapped without affected their functional properties. This strategy enabled the generation of a Kin-Kout channel pair that still exhibits its original function but that shares >90% identity at the protein level. This channel pair will allow fundamental insights into the gating process of potassium channels and may allow to fine-tune these channels types for better adaptation to particular environmental conditions.