Új feszültségfüggő és 2P típusú kálium csatornák klónozása és szerepük vizsgálata = Cloning and functional characterisation of new voltage-dependent and 2P-type

Pályázatunkban feszültségfüggő- és 2P-típusú kálium csatornák klózását, valamint működésük vizsgálatát tűztük ki célul. Elsősorban szabályozásukkal valamint fizológiás szerepükkel foglalkoztunk. A 2P típusú TRESK csatornát másodikként klónoztuk meg, és elsőként írtuk le kalcium-függő szabályozását. Kimutattuk, hogy a TRESK-et ?a 2P háttér csatornák között egyedülálló módon- az intracelluláris kalcium koncentráció emelkedése aktiválja. E hatást egy foszfatáz, a kalcineurin közvetíti. A kalcineurin reverzibilisen defoszforilálja a TRESK csatorna intracelluláris hurok szakaszának 276-os foszfoszerinjét, aminek következtében a csatorna konduktanciája közel 10x-esére fokozódik. Felismertük, hogy a TRESK intracelluláris hurok részén egy olyan szekvencia motívum (PQIVID) van jelen, melyhez hasonló a kalcineurin legismertebb szubsztrátjában, az NFAT-ban is megtalálható. Kimutattuk, hogy a kalciummal aktivált kalcineurin e motívumon keresztül is kapcsolódik a TRESK-hez, ami előfeltétele a csatorna defoszforilációjának/aktiválásának. Vizsgálatainkban részletesen elemeztük a TRESK farmakológiai tulajdonságait, eredményeink alapján a TRESK áram egyrészt elkülönítő más 2P csatornákétól, másrészt megállapítható aktivációjának mértéke is. A Kv8.2 feszültségfüggő kálium csatorna klónozását követően kimutattuk, hogy az alegység elsősorban a retina fotoreceptoraiban expresszálódik, és jelenléte e sejtek számára különleges elektrofiziológiai tulajdonságot biztosít. | The aim of our OTKA project was to clone and characterize 2P-type and voltage dependent potassium channels. We have focused mainly on their regulation and physiological role. We published the second paper about the cloning of the 2P type TRESK channel and we described its calcium dependent regulation. We have shown that TRESK, unlike any other 2P-type potassium channel, is activated by elevation of the cytoplasmic Ca2+ concentration. This effect is mediated by the calcium dependent phosphatase, calcineurin. Calcineurin dephosphorylates phosphoserine 276 in the intracellular loop of TRESK what results in its increased conductance of almost 10-fold. We recognized an amino acid stretch in the intracellular loop of TRESK (PQIVID) which is similar to a motif possessed by the most important substrate of calcineurin, NFAT. We have shown that calcineurin, when activated by Ca2+, attaches to TRESK through this motif and this binding is necessary for the dephosphorylation/activation of the channel. We also analyzed the pharmacological properties of TRESK; based on these results TRESK current can be distinguished from that of other 2P potassium channels. In addition, the degree of the activation of TRESK can also be estimated. After we had cloned the Kv8.2 voltage sensitive potassium channel we showed that the subunit is expressed predominantly in the photoreceptors of the retina and its current significantly contributes to the unique electrophysiological properties of these cells

Formation of Functional Heterodimers by TREK-1 and TREK-2 Two-pore Domain Potassium Channel Subunits.

Two-pore domain (K2P) potassium channels are the major molecular correlates of the background (leak) K+ current in a wide variety of cell types. They generally play a key role in setting the resting membrane potential and regulate the response of excitable cells to various stimuli. K2P channels usually function as homodimers and only a few examples of heteromerization have been previously reported. Expression of the TREK (TWIK-related K+ channel) subfamily members of K2P channels often overlaps in neurons and in other excitable cells. Here we demonstrate that heterologous coexpression of TREK-1 and TREK-2 subunits results in the formation of functional heterodimers. Taking advantage of a tandem construct (in which the two different subunits were linked together to enforce heterodimerization) we characterized the biophysical and pharmacological properties of the TREK-1/TREK-2 current. The heteromer was inhibited by extracellular acidification and by spadin similarly to TREK-1, while its ruthenium red sensitivity was intermediate between TREK-1 and TREK-2 homodimers. The heterodimer has also been distinguished from the homodimers by its unique single channel conductance. Assembly of the two different subunits was confirmed by coimmunoprecipitation of epitope tagged TREK-1 and TREK-2 subunits, coexpressed in Xenopus oocytes. Formation of TREK-1/TREK-2 channels was also demonstrated in native dorsal root ganglion neurons indicating that heterodimerization may provide greater diversity of leak K+ conductances also in native tissues

TRESK Background K+ Channel Is Inhibited by PAR-1/MARK Microtubule Affinity-Regulating Kinases in Xenopus Oocytes

TRESK (TWIK-related spinal cord K+ channel, KCNK18) is a major background K+ channel of sensory neurons. Dominant-negative mutation of TRESK is linked to familial migraine. This important two-pore domain K+ channel is uniquely activated by calcineurin. The calcium/calmodulin-dependent protein phosphatase directly binds to the channel and activates TRESK current several-fold in Xenopus oocytes and HEK293 cells. We have recently shown that the kinase, which is responsible for the basal inhibition of the K+ current, is sensitive to the adaptor protein 14-3-3. Therefore we have examined the effect of the 14-3-3-inhibited PAR-1/MARK, microtubule-associated-protein/microtubule affinity-regulating kinase on TRESK in the Xenopus oocyte expression system. MARK1, MARK2 and MARK3 accelerated the return of TRESK current to the resting state after the calcium-dependent activation. Several other serine-threonine kinase types, generally involved in the modulation of other ion channels, failed to influence TRESK current recovery. MARK2 phosphorylated the primary determinant of regulation, the cluster of three adjacent serine residues (S274, 276 and 279) in the intracellular loop of mouse TRESK. In contrast, serine 264, the 14-3-3-binding site of TRESK, was not phosphorylated by the kinase. Thus MARK2 selectively inhibits TRESK activity via the S274/276/279 cluster, but does not affect the direct recruitment of 14-3-3 to the channel. TRESK is the first example of an ion channel phosphorylated by the dynamically membrane-localized MARK kinases, also known as general determinants of cellular polarity. These results raise the possibility that microtubule dynamics is coupled to the regulation of excitability in the neurons, which express TRESK background potassium channel

Formation of functional heterodimers between the TASK-1 and TASK-3 two-pore domain potassium channel subunits

The potassium channels in the two-pore domain family are widely expressed and regulate the excitability of neurons and other excitable cells. These channels have been shown to function as dimers, but heteromerization between the various channel subunits has not yet been reported. Here we demonstrate that two members of the TASK subfamily of potassium channels, TASK-1 and TASK-3, can form functional heterodimers when expressed in Xenopus laevis oocytes. To recognize the two TASK channel types, we took advantage of the higher sensitivity of TASK-1 over TASK-3 to physiological pH changes and the discriminating sensitivity of TASK-3 to the cationic dye ruthenium red. These features were clearly observed when the channels were expressed individually. However, when TASK-1 and TASK-3 were expressed together, the resulting current showed intermediate pH sensitivity and ruthenium red insensitivity (characteristic of TASK-1), indicating the formation of TASK-1/TASK-3 heterodimers. Expression of a tandem construct in which TASK-3 and TASK-1 were linked together yielded currents with features very similar to those observed when coexpressing the two channels. The tandem construct also responded to AT(1A) angiotensin II receptor stimulation with an inhibition that was weaker than the inhibition of homodimeric TASK-1 and greater than that shown by TASK-3. Expression of epitope-tagged channels in mammalian cells showed their primary presence in the plasma membrane consistent with their function in this location. Heteromerization of two-pore domain potassium channels may provide a greater functional diversity and additional means by which they can be regulated in their native tissues

Mapping aquatic vegetation of the Rakamaz-Tiszanagyfalui Nagy-Morotva using hyperspectral imagery

Rapid development in remote sensing technologies provides more and more reliable methods for environmental assessment. For most wetlands, it is difficult to walk-in without disturbing the endangered species living there; therefore, application of opportunities provided by remote sensing has a great importance in population-mapping. One effective tool of vegetation pattern estimation is hyperspectral remote sensing, which can be used for association and species level mapping as well, due to high ground resolution. The Rakamaz-Tiszanagyfalui Nagy-morotva is an oxbow lake, located in the north-eastern part of Hungary. For this study, a wetland area of 1.17 km2 containing the original water bad and shoreline was selected. For the image analysis, images taken by an AISA DUAL system hyperspectral sensor were used. At the same time, 7 main vegetation classes were separated, which are typical for the sample plot designated on the test site. Classification was performed by the master areas signed by the most common associations of the Rakamaz-Tiszanagyfalui Nagy-morotva with determined spectrums. During the image analysis, SAM classification method was used, where radian values were optimized by the results of classification performed at the control area

Phosphorylation-dependent binding of 14-3-3 proteins controls TRESK regulation

The two-pore domain K+ channel, TRESK (TWIK-related spinal cord K+ channel) is reversibly activated by the calcium/calmodulin-dependent protein phosphatase, calcineurin. In the present study, we report that 14-3-3 proteins directly bind to the intracellular loop of TRESK and control the kinetics of the calcium-dependent regulation of the channel. Coexpression of 14-3-3 eta with TRESK blocked, whereas the coexpression of a dominant negative form of 14-3-3 eta accelerated the return of the K+ current to the resting state after the activation mediated by calcineurin in Xenopus oocytes. The direct action of 14-3-3 was spatially restricted to TRESK, since 14-3-3 eta was also effective, when it was tethered to the channel by a flexible polyglutamine-containing chain. The effect of both the coexpressed and chained 14-3-3 was alleviated by the microinjection of Ser(P)-Raf259 phosphopeptide that competes with TRESK for binding to 14-3-3. The gamma and eta isoforms of 14-3-3 controlled TRESK regulation, whereas the beta, zeta, epsilon, sigma, and tau isoforms failed to influence the mechanism significantly. Phosphorylation of serine 264 in mouse TRESK was required for the binding of 14-3-3 eta. Because 14-3-3 proteins are ubiquitous, they are expected to control the duration of calcineurin-mediated TRESK activation in all the cell types that express the channel, depending on the phosphorylation state of serine 264. This kind of direct control of channel regulation by 14-3-3 is unique within the two-pore domain K+ channel family

TRESK (K2P18.1) Background Potassium Channel is Activated by Novel-Type Protein Kinase C via Dephosphorylation

TRESK (K2P18.1) background K+ channel is a major determinant of the excitability of primary sensory neurons. It has been reported that human TRESK is activated by the protein kinase C (PKC) activator PMA (phorbol 12-myristate 13-acetate) in Xenopus oocytes. In the present study, we investigated the mechanism of this PKC-dependent TRESK regulation. We show that TRESK is activated by the coexpression of the novel-type PKC isoforms eta and epsilon. The effect of PKC is not mediated by calcineurin phosphatase, which is known to evoke the calcium-dependent TRESK activation. The mutations of the calcineurin-binding sites in the channel (PQAAAS-AQAP) did not influence the PMA-induced increase of potassium current. In sharp contrast, the mutations of the target residue of calcineurin in TRESK, S264A and S264E, prevented the effect of PMA. The enforced phosphorylation of S264 by the coexpression of a microtubule-affinity regulating kinase construct (MARK2Δ) also abolished the PKC-dependent TRESK activation. These results suggest that in addition to calcineurin, PKC also regulates TRESK by changing the phosphorylation status of S264. The coexpression of PKC slowed down the recovery of the K+ current to the resting state after the calcineurin-dependent dephosphorylation of TRESK. Therefore, the likely mechanism of action is the PKC-dependent inhibition of the kinase responsible for the (re)phosphorylation of the channel at S264. The PKC-dependent dephosphorylation of TRESK protein was also detected by the Phos-tag SDS-PAGE method. In summary, the activation of novel-type PKC results in the slow (indirect) dephosphorylation of TRESK at the regulatory residue S264 in a calcineurin-independent manner

Chemically Modified Derivatives of the Activator Compound Cloxyquin Exert Inhibitory Effect on TRESK (K2P18.1) Background Potassium Channel.

Cloxyquin has been reported as a specific activator of TRESK (K2P18.1, TWIK-related spinal cord K+ channel) background potassium channel. In this study, we have synthetized chemically modified analogues of cloxyquin and tested their effects on TRESK and other K2P channels. The currents of murine K2P channels, expressed heterologously in Xenopus oocytes, were measured by two-electrode voltage clamp, whereas the native background K+ conductance of mouse dorsal root ganglion (DRG) neurons was examined by the whole-cell patch clamp method. Some of the analogues retained the activator character of the parent compound, but more interestingly, other derivatives inhibited mouse TRESK current. The inhibitor analogues (A2764 and A2793) exerted state-dependent effect. The degree of inhibition by 100 µM A2764 (77.8±1.5%, n=6) was larger in the activated state of TRESK (i.e. after calcineurin-dependent stimulation) than in the resting state of the channel (42.8±4.3% inhibition, n=7). The selectivity of the inhibitor compounds was tested on several K2P channels. A2793 inhibited TASK-1 (100 µM, 53.4±6%, n=5), while A2764 was more selective for TRESK, it only moderately influenced TREK-1 and TALK-1. The effect of A2764 was also examined on the background K+ currents of DRG neurons. A subpopulation of DRG neurons, prepared from wild-type animals, expressed background K+ currents sensitive to A2764, while the inhibitor did not affect the currents in the DRG neurons of TRESK-deficient mice. Accordingly, A2764 may prove to be useful for the identification of TRESK current in native cells, and for the investigation of the role of the channel in nociception and migraine