Clustering of the K+ channel GORK of Arabidopsis parallels its gating by extracellular K+

GORK is the only outward‐rectifying Kv‐like K⁺ channel expressed in guard cells. Its activity is tightly regulated to facilitate K⁺ efflux for stomatal closure and is elevated in ABA in parallel with suppression of the activity of the inward‐rectifying K⁺ channel KAT1. Whereas the population of KAT1 is subject to regulated traffic to and from the plasma membrane, nothing is known about GORK, its distribution and traffic in vivo. We have used transformations with fluorescently‐tagged GORK to explore its characteristics in tobacco epidermis and Arabidopsis guard cells. These studies showed that GORK assembles in puncta that reversibly dissociated as a function of the external K⁺ concentration. Puncta dissociation parallelled the gating dependence of GORK, the speed of response consistent with the rapidity of channel gating response to changes in the external ionic conditions. Dissociation was also suppressed by the K⁺ channel blocker Ba²⁺. By contrast, confocal and protein biochemical analysis failed to uncover substantial exo‐ and endocytotic traffic of the channel. Gating of GORK is displaced to more positive voltages with external K⁺, a characteristic that ensures the channel facilitates only K⁺ efflux regardless of the external cation concentration. GORK conductance is also enhanced by external K⁺ above 1 mm . We suggest that GORK clustering in puncta is related to its gating and conductance, and reflects associated conformational changes and (de)stabilisation of the channel protein, possibly as a platform for transmission and coordination of channel gating in response to external K⁺.     

The S1–S2 linker determines the distinct pH sensitivity between ZmK2.1 and KAT1

Efficient stomatal opening requires activation of KAT‐type K⁺ channels, which mediate K⁺ influx into guard cells. Most KAT‐type channels are functionally facilitated by extracellular acidification. However, despite sequence and structural homologies, the maize counterpart of Arabidopsis KAT1 (ZmK2.1) is resistant to pH activation. To understand the structural determinant that results in the differential pH activation of these counterparts, we analysed chimeric channels and channels with point mutations for ZmK2.1 and its closest Arabidopsis homologue KAT1. Exchange of the S1–S2 linkers altered the pH sensitivity between the two channels, suggesting that the S1–S2 linker is essentially involved in the pH sensitivity. The effects of D92 mutation within the linker motif together with substitution of the first half of the linker largely resemble the effects of substitution of the complete linker. Topological modelling predicts that one of the two cysteines located on the outer face section of the S5 domain may serve as a potential titratable group that interacts with the S1–S2 linker. The difference between ZmK2.1 and KAT1 is predicted to be the result of the distance of the stabilized linkers from the titratable group. In KAT1, residue K85 within the linker forms a hydrogen bond with C211 that enables the pH activation; conversely, the linker of ZmK2.1 is distantly located and thus does not interact with the equivalent titration group (C208). Thus, in addition to the known structural contributors to the proton activation of KAT channels, we have uncovered a previously unidentified component that is strongly involved in this complex proton activation network.     

Single mutations strongly alter the K-selective pore of the Kin channel KAT1

Voltage-dependent potassium uptake channels represent the major pathway for K⁺ accumulation underlying guard cell swelling and stomatal opening. The core structure of these Shaker-like channels is represented by six transmembrane domains and an amphiphilic pore-forming region between the fifth and sixth domain. To explore the effect of point mutations within the stretch of amino acids lining the K⁺ conducting pore of KAT1, an Arabidopsis thaliana guard cell K_in channel, we selected residues deep inside and in the periphery of the pore. The mutations on positions 256 and 267 strongly altered the interaction of the permeation pathway with external Ca²⁺ ions. Point mutations on position 256 in KAT1 affected the affinity towards Ca²⁺, the voltage dependence as well as kinetics of the Ca²⁺ blocking reaction. Among these T256S showed a Ca²⁺ phenotype reminiscent of an inactivation-like process, a phenomenon unknown for K_in channels so far. Mutating histidine 267 to alanine, a substitution strongly affecting C-type inactivation in Shaker, this apparent inactivation could be linked to a very slow calcium block. The mutation H267A did not affect gating but hastened the Ca²⁺ block/unblock kinetics and increased the Ca²⁺ affinity of KAT1. From the analysis of the presented data we conclude that even moderate point mutations in the pore of KAT1 seem to affect the pore geometry rather than channel gating.     

Structure and function of potassium channels in plants: some inferences about the molecular origin of inward rectification in KAT1 channels (Review)

Potassium channels in plants play a variety of important physiological roles including K⁺ uptake into roots, stomatal and leaf movements, and release of K⁺ into the xylem. This review summarizes current knowledge about a class of plant genes whose products are K⁺ channel-forming proteins. Potassium channels of this class belong to a superfamily characterized by six membrane-spanning domains (S1-6), a positively charged S4 domain and a region between the S5 and S6 segments that forms the channel selectivity filter. These channels are voltage dependent, which means the membrane potential modifies the probability of opening (P_o). However, despite these channels sharing the same topology as the outward-rectifying K⁺ channels, which are activated by membrane depolarization, some plant K⁺ channels such as KAT1/2 and KST1 open with hyperpolarizing voltages. In outward-rectifying K⁺ channels, the change in P_o is achieved through a voltage sensor formed by the S4 segment that detects the voltage transferring its energy to the gate that controls pore opening. This coupling is achieved by an outward displacement of the charges contained in S4. In KAT1, most of the results indicate that S4 is the voltage sensor. However, how the movement of S4 leads to opening remains unanswered. On the basis of recent data, we propose here that in plant-inward rectifiers an inward movement of S4 leads to channel opening and that the difference between it and outward-rectifying channels resides in the mechanism that couples gating charge displacement with pore opening.     

Evidence for a Multi-ion Pore Behavior in the Plant Potassium Channel KAT1

KAT1 is a cloned voltage-gated K⁺ channel from the plant Arabidopsis thaliana L., which displays an inward rectification reminiscent of `anomalous' rectification of the i <sub>f</sub> pacemaker current recorded in animal cells. Macroscopic conductance of KAT1 expressed in Xenopus oocytes was 5-fold less in pure Rb<sup>+</sup> solution than in pure K<sup>+</sup> solution, and negligible in pure Na<sup>+</sup> solution. Experiments in different K<sup>+</sup>/Na<sup>+</sup> or K<sup>+</sup>/Rb<sup>+</sup> mixtures revealed deviations from the principle of independence and notably two anomalous effects of the K<sup>+</sup>/Rb<sup>+</sup> mole fraction (i.e., the ratio [K<sup>+</sup>]/([K<sup>+</sup>]+[Rb<sup>+</sup>])). First, the KAT1 deactivation time constant was both voltage- and mole fraction-dependent (a so-called `foot in the door' effect was thus observed in KAT1 channel). Second, when plotted against the K⁺/Rb⁺ mole fraction, KAT1 conductance values passed through a minimum. This minimum is more important for two pore mutants of KAT1 (T259S and T260S) that displayed an increase in P_Rb/P_K. These results are consistent with the idea that KAT1 conduction requires several ions to be present simultaneously within the pore. Therefore, this atypical `green' member of the Shaker superfamily of K⁺ channels further shows itself to be an interesting model as well for permeation as for gating mechanism studies. Received: 9 February 1998/Revised: 28 July 1998     

Voltage-dependent Gating of Single Wild-Type and S4 Mutant KAT1 Inward Rectifier Potassium Channels

The voltage-dependent gating mechanism of KAT1 inward rectifier potassium channels was studied using single channel current recordings from Xenopus oocytes injected with KAT1 mRNA. The inward rectification properties of KAT1 result from an intrinsic gating mechanism in the KAT1 channel protein, not from pore block by an extrinsic cation species. KAT1 channels activate with hyperpolarizing potentials from −110 through −190 mV with a slow voltage-dependent time course. Transitions before first opening are voltage dependent and account for much of the voltage dependence of activation, while transitions after first opening are only slightly voltage dependent. Using burst analysis, transitions near the open state were analyzed in detail. A kinetic model with multiple closed states before first opening, a single open state, a single closed state after first opening, and a closed-state inactivation pathway accurately describes the single channel and macroscopic data. Two mutations neutralizing charged residues in the S4 region (R177Q and R176L) were introduced, and their effects on single channel gating properties were examined. Both mutations resulted in depolarizing shifts in the steady state conductance–voltage relationship, shortened first latencies to opening, decreased probability of terminating bursts, and increased burst durations. These effects on gating were well described by changes in the rate constants in the kinetic model describing KAT1 channel gating. All transitions before the open state were affected by the mutations, while the transitions after the open state were unaffected, implying that the S4 region contributes to the early steps in gating for KAT1 channels.     

Orientation of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> KAT1 Channel in the Plasma Membrane

The Arabidopsis thaliana KAT1, an inward-rectifying potassium channel, shares molecular features with the Shaker family of outward rectifier K⁺ channels. The KAT1 amino-acid sequence reveals the presence of a positively charged S4 and a segment containing the TXGYGD signature sequence in the pore (P) region. To test whether the inward-rectifying properties of KAT1 are due to reverse orientation in the membrane, such that the voltage sensor is oriented in the opposite direction of the electric field compared with the Shaker K⁺ channel, we have inserted a flag epitope in the NH₂ terminus or the S3–S4 loop. The KAT1 and tagged constructs expressed functional channels in whole cells, Xenopus oocytes and COS-7. The electrophysiological properties of both tagged constructs were similar to those of the wild type. Immunofluorescence with an antibody against the flag epitope and an anti-C terminal KAT1 determined the membrane localization of these epitopes and the orientation of the KAT1 channel in the membrane. Our data confirm that KAT1 in eukaryotic cells has an orientation similar to the Shaker K⁺ channel.     

External Cd2+ and protons activate the hyperpolarization-gated K+ channel KAT1 at the voltage sensor

The functionally diverse cyclic nucleotide binding domain (CNBD) superfamily of cation channels contains both depolarization-gated (e.g., metazoan EAG family K⁺ channels) and hyperpolarization-gated channels (e.g., metazoan HCN pacemaker cation channels and the plant K⁺ channel KAT1). In both types of CNBD channels, the S4 transmembrane helix of the voltage sensor domain (VSD) moves outward in response to depolarization. This movement opens depolarization-gated channels and closes hyperpolarization-gated channels. External divalent cations and protons prevent or slow movement of S4 by binding to a cluster of acidic charges on the S2 and S3 transmembrane domains of the VSD and therefore inhibit activation of EAG family channels. However, a similar divalent ion/proton binding pocket has not been described for hyperpolarization-gated CNBD family channels. We examined the effects of external Cd²⁺ and protons on Arabidopsis thaliana KAT1 expressed in Xenopus oocytes and found that these ions strongly potentiate voltage activation. Cd²⁺ at 300 µM depolarizes the V₅₀ of KAT1 by 150 mV, while acidification from pH 7.0 to 4.0 depolarizes the V₅₀ by 49 mV. Regulation of KAT1 by Cd²⁺ is state dependent and consistent with Cd²⁺ binding to an S4-down state of the VSD. Neutralization of a conserved acidic charge in the S2 helix in KAT1 (D95N) eliminates Cd²⁺ and pH sensitivity. Conversely, introduction of acidic residues into KAT1 at additional S2 and S3 cluster positions that are charged in EAG family channels (N99D and Q149E in KAT1) decreases Cd²⁺ sensitivity and increases proton potentiation. These results suggest that KAT1, and presumably other hyperpolarization-gated plant CNBD channels, can open from an S4-down VSD conformation homologous to the divalent/proton-inhibited conformation of EAG family K⁺ channels.     

The K+ channel KZM2 is involved in stomatal movement by modulating inward K+ currents in maize guard cells

Stomata are the major gates in plant leaf that allow water and gas exchange, which is essential for plant transpiration and photosynthesis. Stomatal movement is mainly controlled by the ion channels and transporters in guard cells. In Arabidopsis, the inward Shaker K⁺ channels, such as KAT1 and KAT2, are responsible for stomatal opening. However, the characterization of inward K⁺ channels in maize guard cells is limited. In the present study, we identified two KAT1‐like Shaker K⁺ channels, KZM2 and KZM3, which were highly expressed in maize guard cells. Subcellular analysis indicated that KZM2 and KZM3 can localize at the plasma membrane. Electrophysiological characterization in HEK293 cells revealed that both KZM2 and KZM3 were inward K⁺ (K_in) channels, but showing distinct channel kinetics. When expressed in Xenopus oocytes, only KZM3, but not KZM2, can mediate inward K⁺ currents. However, KZM2 can interact with KZM3 forming heteromeric K_in channel. In oocytes, KZM2 inhibited KZM3 channel conductance and negatively shifted the voltage dependence of KZM3. The activation of KZM2–KZM3 heteromeric channel became slower than the KZM3 channel. Patch‐clamping results showed that the inward K⁺ currents of maize guard cells were significantly increased in the KZM2 RNAi lines. In addition, the RNAi lines exhibited faster stomatal opening after light exposure. In conclusion, the presented results demonstrate that KZM2 functions as a negative regulator to modulate the K_in channels in maize guard cells. KZM2 and KZM3 may form heteromeric K_in channel and control stomatal opening in maize.     

Increased Resistance to Extracellular Cation Block by Mutation of the Pore Domain of the Arabidopsis Inward-rectifying K+ Channel KAT1

Inward-rectifying potassium channels in plant cells provide important mechanisms for low-affinity K⁺ uptake and membrane potential control in specific cell types, including guard cells, pulvinus cells, aleurone cells and root hair cells. K⁺ channel blockers are potent tools for studying the physiological functions and structural properties of K⁺ channels. In the present study the structural and biophysical mechanisms of Cs⁺ and TEA⁺ block of a cloned Arabidopsis inward-rectifying K⁺ channel (KAT1) were analyzed. Effects of the channel blockers Cs⁺ and TEA⁺ were characterized both extracellularly and intracellularly. Both external Cs⁺ and TEA⁺ block KAT1 currents. A mutant of KAT1 (``m2KAT1'; H267T, E269V) was produced by site-directed mutagenesis of two amino acid residues in the C-terminal portion of the putative pore (P) domain. This mutant channel was blocked less by external Cs⁺ and TEA⁺ than the wild-type K⁺ channel. Internal TEA⁺ and Cs⁺ did not significantly block either m2KAT1 or KAT1 channels. Other properties, such as cation selectivity, voltage-dependence and proton activation did not show large changes between m2KAT1 and KAT1, demonstrating the specificity of the introduced mutations. These data suggest that the amino acid positions mutated in the inward-rectifying K⁺ channel, KAT1, are accessible to external blockers and may be located on the external side of the membrane, as has been suggested for outward-rectifying K⁺ channels. Received: 31 July 1995/Revised: 5 January 1996     

Differential regulation of K+ channels in Arabidopsis epidermal and stelar root cells

The patch clamp technique was applied to protoplasts isolated from the epidermis and pericycle of Arabidopsis roots and their plasma membrane currents investigated. In the whole cell configuration, all protoplasts from the epidermis exhibited depolarization‐activated time‐dependent outwardly rectifying (OR) currents whereas OR currents were present in only 50% of cells from the pericycle. The properties of the OR currents in the epidermis and pericycle were compared with respect to their selectivity, pharmacology and gating. The time‐dependent activation kinetics, selectivity and sensitivity to extracellular tetraethyl ammonium of the OR current in each cell type were not significantly different. The reversal potential (E_rev) of the OR currents indicated that they were primarily due to the movement of K⁺. However, the gating properties of the OR currents from the epidermis differed markedly from those exhibited in the pericycle. Although both cell types displayed OR currents with voltage‐dependent gating modulated in a potassium‐dependent fashion [i.e. the activation threshold (V_0.5) was displaced to more positive voltages as extracellular K⁺ increased], the OR currents in the epidermis also displayed voltage‐independent gating by extracellular K⁺ which dramatically regulated current density. In the present study, reducing extracellular K⁺ activity from 40 to 0.87 mm reduced the OR current density in epidermal cells by approximately 80%. The chord conductance of the OR current saturated as a function of extracellular K⁺ and could be fitted with a Michaelis–Menten function to yield a binding constant (K_m) of 10.5 mm . The ability of other monovalent cations to substitute for K⁺‐gating of the OR currents was also investigated and shown to exhibit a relative sequence of K⁺ ≥ Rb⁺ > Cs⁺ > Na⁺ ≥ Li⁺ (Eisenmann sequence IV) with respect to efficacy of gating. Furthermore, single channel recordings demonstrated that channel activity rather than the single channel conductance was modulated by extracellular K⁺. In contrast, OR current density in the pericycle was largely independent of extracellular K⁺. It is suggested that the contrasting gating properties of the K⁺ channels in the epidermis and pericycle reflect their different physiological roles, particularly with respect to their role in K⁺ (nutrient) transport from the soil solution to the shoot.     

Coupling of activation and inactivation gate in a K+‐channel: potassium and ligand sensitivity

Potassium (K⁺)‐channel gating is choreographed by a complex interplay between external stimuli, K⁺ concentration and lipidic environment. We combined solid‐state NMR and electrophysiological experiments on a chimeric KcsA–Kv1.3 channel to delineate K⁺, pH and blocker effects on channel structure and function in a membrane setting. Our data show that pH‐induced activation is correlated with protonation of glutamate residues at or near the activation gate. Moreover, K⁺ and channel blockers distinctly affect the open probability of both the inactivation gate comprising the selectivity filter of the channel and the activation gate. The results indicate that the two gates are coupled and that effects of the permeant K⁺ ion on the inactivation gate modulate activation‐gate opening. Our data suggest a mechanism for controlling coordinated and sequential opening and closing of activation and inactivation gates in the K⁺‐channel pore.     

Isolation and characterization of SsmTx‐I,a Specific Kv2.1 blocker from the venom of the centipede Scolopendra Subspinipes Mutilans L. Koch

Scolopendra subspinipes mutilans, also known as Chinese red‐headed centipede, is a venomous centipede from East Asia and Australasia. Venom from this animal has not been researched as thoroughly as venom from snakes, snails, scorpions, and spiders. In this study, we isolated and characterized SsmTx‐I, a novel neurotoxin from the venom of S. subspinipes mutilans. SsmTx‐I contains 36 residues with four cysteines forming two disulfide bonds. It had low sequence similarity (<10%) with other identified peptide toxins. By whole‐cell recording, SsmTx‐I significantly blocked voltage‐gated K⁺ channels in dorsal root ganglion neurons with an IC₅₀ value of 200 nM, but it had no effect on voltage‐gated Na⁺ channels. Among the nine K⁺ channel subtypes expressed in human embryonic kidney 293 cells, SsmTx‐I selectively blocked the Kv2.1 current with an IC₅₀ value of 41.7 nM, but it had little effect on currents mediated by other K⁺ channel subtypes. Blockage of Kv2.1 by SsmTx‐I was not associated with significant alteration of steady‐state activation, suggesting that SsmTx‐I might act as a simple inhibitor or channel blocker rather than a gating modifier. Our study reported a specific Kv2.1‐blocker from centipede venom and provided a basis for future investigations of SsmTx‐I, for example on structure–function relationships, mechanism of action, and pharmacological potential. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Pronounced differences between the native K+ channels and KAT1 and KST1 α-subunit homomers of guard cells

Stomatal opening is the result of K⁺-salt accumulation in guard cells. Potassium uptake in these motor cells is mediated by voltage-dependent, K⁺-selective ion channels. Here we compare the in-vitro properties of two guard-cell K⁺-channel α-subunits from Arabidopsis thaliana (L.) Heynh. (KAT1) and Solanum tuberosum L. (KST1) after heterologous expression with the respective K⁺-transport characteristics in their mother cell. The KAT1 and KST1 subunits when expressed in Xenopus oocytes shared the basic features of the K⁺-uptake channels in the corresponding guard cells, including voltage dependence and single-channel conductance. Besides these similarities, the electrophysiological comparison of K⁺ channels in the homologous and the heterologous expression systems revealed pronounced differences with respect to modulation and block by extracellular cations. In the presence of 1 mM Cs⁺, 50% of the guard-cell K⁺-uptake channels (GCKC1_in) in A. thaliana and S. tuberosum, were inhibited upon hyperpolarization to −90 mV. For a similar effect on KAT1 and KST1 in oocytes, voltages as negative as −155 mV were required. In contrast, compared to the K⁺ channels in vivo the functional α-subunit homomers almost lacked a voltage-dependent block by extracellular Ca²⁺. Similar to the block by Cs⁺ and Ca²⁺, the acid activation of the α-homomers was less pronounced in oocytes. Upon acidification the voltage-dependence shifted by 82 and 90 mV for GCKCL_in in A. thaliana and S. tuberosum, respectively, but only by 25 mV for KAT1 and KST1. From the differences in K⁺-channel modulation in vivo and after heterologous expression we conclude that the properties of functional guard-cell K⁺-uptake channels result either from the heterometric assembly of different α-subunits or evolve from cell-type-specific posttranslational modification. Received: 6 March 1998 / Accepted: 9 July 1998     

Suppression of Inward-Rectifying K+ Channels KAT1 and AKT2 by Dominant Negative Point Mutations in the KAT1 α-Subunit

The Arabidopsis thaliana cDNA, KAT1 encodes a hyperpolarization-activated K⁺ (K⁺ _in ) channel. In the present study, we identify and characterize dominant negative point mutations that suppress K⁺ _in channel function. Effects of two mutations located in the H5 region of KAT1, at positions 256 (T256R) and 262 (G262K), were studied. The co-expression of either T256R or G262K mutants with KAT1 produced an inhibition of K⁺ currents upon membrane hyperpolarization. The magnitude of this inhibition was dependent upon the molar ratio of cRNA for wild-type to mutant channel subunits injected. Inhibition of KAT1 currents by the co-expression of T256R or G262K did not greatly affect the ion selectivity of residual currents for Rb⁺, Na⁺, Li⁺, or Cs⁺. When T256R or G262K were co-expressed with a different K⁺ channel, AKT2, an inhibition of the channel currents was also observed. Voltage-dependent Cs⁺ block experiments with co-expressed wild type, KAT1 and AKT2, channels further indicated that KAT1 and AKT2 formed heteromultimers. These data show that AKT2 and KAT1 are able to co-assemble and suggest that suppression of channel function can be pursued in vivo by the expression of the dominant negative K ⁺ _in channel mutants described here. Received: 2 July 1998/Revised: 23 October 1998     

Role of hydrophobic and ionic forces in the movement of S4 of the Shaker potassium channel

Abstract

Voltage-gated ion (K⁺, Na⁺, Ca²⁺) channels contain a pore domain (PD) surrounded by four voltage sensing domains (VSD). Each VSD is made up of four transmembrane helices, S1–S4. S4 contains 6–7 positively charged residues (arginine/lysine) separated two hydrophobic residues, whereas S1–S3 contribute to two negatively charged clusters. These structures are conserved among all members of the voltage-gated ion channel family and play essential roles in voltage gating. The role of S4 charged residues in voltage gating is well established: During depolarization, they move out of the membrane electric field, exerting a mechanical force on channel gates, causing them to open. However, the role of the intervening hydrophobic residues in voltage sensing is unclear. Here we studied the role of these residues in the prototypical Shaker potassium channel. We have altered the physicochemical properties of both charged and hydrophobic positions of S4 and examined the effect of these modifications on the gating properties of the channel. For this, we have introduced cysteines at each of these positions, expressed the mutants in Xenopus oocytes, and examined the effect of in situ addition of charge, via Cd²⁺, on channel gating by two-electrode voltage clamp. Our results reveal a face of the S4 helix (comprising residues L358, L361, R365 and R368) where introduction of charge at hydrophobic positions destabilises the closed state and removal of charges from charged positions has an opposite effect. We propose that hydrophobic residues play a crucial role in limiting gating to a physiological voltage range.     

Phosphatidic acid directly binds with rice potassium channel OsAKT2 to inhibit its activity

The plant Shaker K⁺ channel AtAKT2 has been identified as a weakly rectifying channel that can stabilize membrane potentials to promote photoassimilate phloem loading and translocation. Thus, studies on functional characterization and regulatory mechanisms of AtAKT2‐like channels in crops are highly important for improving crop production. Here, we identified the rice OsAKT2 as the ortholog of Arabidopsis AtAKT2, which is primarily expressed in the shoot phloem and localized at the plasma membrane. Using an electrophysiological assay, we found that OsAKT2 operated as a weakly rectifying K⁺ channel, preventing H⁺/sucrose‐symport‐induced membrane depolarization. Three critical amino acid residues (K193, N206, and S326) are essential to the phosphorylation‐mediated gating change of OsAKT2, consistent with the roles of the corresponding sites in AtAKT2. Disruption of OsAKT2 results in delayed growth of rice seedlings under short‐day conditions. Interestingly, the lipid second messenger phosphatidic acid (PA) inhibits OsAKT2‐mediated currents (both instantaneous and time‐dependent components). Lipid dot‐blot assay and liposome‐protein binding analysis revealed that PA directly bound with two adjacent arginine residues in the ANK domain of OsAKT2, which is essential to PA‐mediated inhibition of OsAKT2. Electrophysiological and phenotypic analyses also showed the PA‐mediated inhibition of AtAKT2 and the negative correlation between intrinsic PA level and Arabidopsis growth, suggesting that PA may inhibit AKT2 function to affect plant growth and development. Our results functionally characterize the Shaker K⁺ channel OsAKT2 and reveal a direct link between phospholipid signaling and plant K⁺ channel modulation.     

Cooperative nature of gating transitions in K+ channels as seen from dynamic importance sampling calculations

The growing dataset of K⁺ channel x‐ray structures provides an excellent opportunity to begin a detailed molecular understanding of voltage‐dependent gating. These structures, while differing in sequence, represent either a stable open or closed state. However, an understanding of the molecular details of gating will require models for the transitions and experimentally testable predictions for the gating transition. To explore these ideas, we apply dynamic importance sampling to a set of homology models for the molecular conformations of K⁺ channels for four different sets of sequences and eight different states. In our results, we highlight the importance of particular residues upstream from the Pro‐Val‐Pro (PVP) region to the gating transition. This supports growing evidence that the PVP region is important for influencing the flexibility of the S6 helix and thus the opening of the gating domain. The results further suggest how gating on the molecular level depends on intra‐subunit motions to influence the cooperative behavior of all four subunits of the K⁺ channel. We hypothesize that the gating process occurs in steps: first sidechain movement, then inter‐S5‐S6 subunit motions, and lastly the large‐scale domain rearrangements. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

ATP-sensitive potassium channels and vascular function

Fluorescence-based approaches provide powerful techniques to directly report structural dynamics underlying gating processes in Shaker K_V channels. Here, following on from work carried out in Shaker channels, we have used voltage clamp fluorimetry for the first time to study voltage sensor motions in mammalian K_V1.5 channels, by attaching TMRM fluorescent probes to substituted cysteine residues in the S3-S4 linker of K_V1.5 (A397C). Compared with the Shaker channel, there are significant differences in the fluorescence signals that occur on activation of the channel. In addition to a well-understood fluorescence quenching signal associated with S4 movement, we have recorded a unique partial recovery of fluorescence after the quenching that is attributable to gating events at the outer pore mouth,¹ that is not seen in Shaker despite significant homology between it and Kv1.5 channels in the S5-P loop-S6 region. Extracellular potassium is known to modulate C-type inactivation in Shaker and K_V channels at sites in the outer pore mouth, and so here we have measured the concentration-dependence of potassium effects on the fluorescence recovery signals from A397C. Elevation of extracellular K⁺ inhibits the rapid fluorescence recovery, with complete abolition at 99 mM K⁺, and an IC₅₀ of 29 mM K⁺_o. These experiments suggest that the rapid fluorescence recovery reflects early gating movements associated with inactivation, modulated by extracellular K⁺, and further support the idea that outer pore motions occur rapidly after K_V1.5 channel opening and can be observed by fluorophores attached to the S3-S4 linker.