2P Channels Research Articles

Two-pore potassium channels in the cardiovascular system

Two-pore domain (K(2P)) channels emerged about a decade ago and since then have been an expanding area of interest. This is because their biophysical and pharmacological properties make them good candidates to support background potassium currents and membrane potential in many cell types. There is clear evidence for TREK-1 and TASK-1 in the heart and these channels are likely to regulate cardiac action potential duration through their regulation by stretch, polyunsaturated fatty acids, pH, and neurotransmitters. TREK-1 may also have a critical role in mediating the vasodilator response of resistance arteries to polyunsaturated fatty acids, thus contributing to their protective effect on the cardiovascular system. TASK-1, on the other hand, is a strong candidate for a role in hypoxic vasoconstriction of pulmonary arteries. Many other members of the K(2P) channel family have been identified in the cardiovascular system, although their functional roles are still to be demonstrated. This review provides an up to date summary of what is known about the involvement of members of the K(2P) channel family in cells of the heart and arterial circulation. Our knowledge of their roles will improve with the rapidly increasing interest in them and as new selective pharmacological tools emerge. As their physiological roles emerge, the K(2P) family of potassium channels may offer promising therapeutic solutions to target cardiovascular diseases.

Fourth Conference on Epileptogenesis, May 23–26, 2007, Pisa, Italy

Fourth Conference on Epileptogenesis, May 23–26, 2007, Pisa, Italy

Functional consequences of leucine and tyrosine mutations in the dual pore motifs of the yeast K+ channel, Tok1p

Tandem pore-loop potassium channels differ from the majority of K(+) channels in that a single polypeptide chain carries two K(+)-specific segments (P) each sandwiched between two transmembrane helices (M) to form an MP(1)M-MP(2)M series. Two of these peptide molecules assemble to form one functional potassium channel, which is expected to have biaxial symmetry (commonly described as asymmetric) due to independent mutation in the two MPM units. The resulting intrinsic asymmetry is exaggerated in fungal 2P channels, especially in Tok1p of Saccharomyces, by the N-terminal presence of four more transmembrane helices. Functional implications of such structural asymmetry have been investigated via mutagenesis of residues (L290 in P(1) and Y424 in P(2)) that are believed to provide the outermost ring of carbonyl oxygen atoms for coordination with potassium ions. Both complementary mutations (L290Y and Y424L) yield functional potassium channels having quasi-normal conductance when expressed in Saccharomyces itself, but the P(1) mutation (only) accelerates channel opening about threefold in response to depolarizing voltage shifts. The more pronounced effect at P(1) than at P(2) appears paradoxical in relation to evolution, because a comparison of fungal Tok1p sequences (from 28 ascomycetes) shows the filter sequence of P(2) (overwhelmingly TIGYGD) to be much stabler than that of P(1) (mostly TIGLGD). Profound functional asymmetry is revealed by the fact that combining mutations (L290Y + Y424L)-which inverts the order of residues from the wild-type channel-reduces the expressed channel conductance by a large factor (20-fold, cf. <twofold for the single mutants).

TRESK two-pore-domain K+ channels constitute a significant component of background potassium currents in murine dorsal root ganglion neurones.

TRESK (TWIK-related spinal cord K(+) channel) is the most recently identified member of the two-pore-domain potassium channel (K(2P)) family, the molecular source of background potassium currents. Human TRESK channels are not affected by external acidification. However, the mouse orthologue displays moderate pH dependence isolated to a single histidine residue adjacent to the GYG selectivity filter. In the human protein, sequence substitution of tyrosine by histidine at this critical position generated a mutant that displays almost identical proton sensitivity compared with mouse TRESK. In contrast to human TRESK, which is specifically located in spinal cord, we detected mouse TRESK (mTRESK) mRNA in several epithelial and neuronal tissues including lung, liver, kidney, brain and spinal cord. As revealed by endpoint and quantitative RT-PCR, mTRESK channels are mainly expressed in dorsal root ganglia (DRG) and on the transcript level represent the most important background potassium channel in this tissue. DRG neurones of all sizes were labelled by in situ hybridizations with TRESK-specific probes. In DRG neurones of TRESK[G339R] functional knock-out (KO) mice the standing outward current IK(so) was significantly reduced compared with TRESK wild-type (WT) littermates. Different responses to K(2P) channel regulators such as bupivacaine, extracellular protons and quinidine corroborated the finding that approximately 20% of IK(so) is carried by TRESK channels. Unexpectedly, we found no difference in resting membrane potential between DRG neurones of TRESK[WT] and TRESK[G339R] functional KO mice. However, in current-clamp recordings we observed significant changes in action potential duration and amplitude of after-hyperpolarization. Most strikingly, cellular excitability of DRG neurones from functional KO mice was significantly augmented as revealed by reduced rheobase current to elicit action potentials.

Expression of thermosensitive two‐pore domain K+ channels in human keratinocytes cell line HaCaT cells

Recent studies have shown that keratinocytes can sense temperature via thermo-transient receptor potential (TRP) channels. It is not known whether other thermosensitive ion channels such as TREK-1, TREK-2 and TRAAK (TREKs/TRAAK) that are members of the two-pore domain K(+) (K(2P)) channel family are expressed in human keratinocytes. Here, we identified the expression of TREKs/TRAAK in human keratinocytes-derived cell line HaCaT cells using RT-PCR, immunocytochemistry, Western blot analysis and patch-clamp technique. RT-PCR showed that all six K(2P) channels tested (TASK-1, TASK-3, TREK-1, TREK-2, TRAAK and TASK-2) were expressed in HaCaT cells, as well as in skin and dorsal root ganglion (DRG) of rat. The expression of TREKs/TRAAK mRNA identified by RT-PCR was further studied at the protein level. Using anti-TREK-1, -TREK-2 and -TRAAK, bands of approximately 46, approximately 60 and approximately 43 kDa, respectively, were observed at plasma membrane of HaCaT cells. Immunostaining also showed that TREK-1, TREK-2 and TRAAK were expressed in all area of cells including plasma membrane. Whole-cell K(+) currents recorded from HaCaT cells were activated by arachidonic acid and heat. These results suggest that TREKs/TRAAK channels could act as thermosensors in human keratinocytes.

MGlu4 potentiation of K 2P2.1 is dependant on C-terminal dephosphorylation

Two-pore domain potassium (K 2P) channels are proposed to underlie the background or leak current found in many excitable cells. Extensive studies have been performed investigating the inhibition of K 2P2.1 by Gα q- and Gα s-coupled G-protein-coupled receptors (GPCRs), whereas in the present study we investigate the mechanisms underlying Gα i/Gα o-coupled GPCR increases in K 2P2.1 activity. Activation of mGlu4 increases K 2P2.1 activity, with pharmacological inhibition of protein kinases and phosphatases revealing the involvement of PKA whereas PKC, PKG or protein phosphatases play no role. Mutational analysis of potential C-terminal phosphorylation sites indicates S333 to control approximately 70%, with S300 controlling approximately 30% of the increase in K 2P2.1 activity following mGlu4 activation. These data reveal that activation of mGlu4 leads to an increase in K 2P2.1 activity through a reduction in C-terminal phosphorylation, which represents a novel mechanism by which group III mGlu receptors may regulate cell excitability and synaptic activity.

Starring TREK-1

See related article, pages 176–184 “We are more alike than unlike, my dear Captain. I have pores, humans have pores.” Lieutenant Commander Data, Stardate 41209.2 Before 1996, all known mammalian K+ channels were classified into only two different structural families according to the number of transmembrane (TM) spanning and pore-forming (P) domains in their α subunit. One family is characterized by K+ channels composed of two TM domains and one P domain, and includes the inwardly rectifying and ATP-sensitive K+ channels. The second family represents K+ channels characterized by six or seven TM domains and one P domain, and includes the voltage-gated and Ca2+-activated K+ channels. To form one functional channel for either of these families, four α subunits assemble to establish one K+ permeable pore. In the mid 1990s, researchers took advantage of the fact that the P domain of each K+ channel’s pore-forming α subunit is highly conserved across species and represents a common structural motif.1 Genome searches for DNA sequences coding for the P domain revealed a unique K+ channel in yeast and Caenorhabditis elegans that contained two P domains within a single α subunit polypeptide having eight potential TM domains.2 The following year a human K+ channel was cloned that also showed the unique feature of two P domains, but displayed four TM domains in a single α subunit (Figure 1).3 The channel was given the name TWIK-1 ( T andem of P domains in a W eak I nward rectifying K + channel). Since the cloning of TWIK-1, a total of fifteen …

The neuronal background K2P channels: focus on TREK1

Two-pore-domain K(+) (K(2P)) channel subunits are made up of four transmembrane segments and two pore-forming domains that are arranged in tandem and function as either homo- or heterodimeric channels. This structural motif is associated with unusual gating properties, including background channel activity and sensitivity to membrane stretch. Moreover, K(2P) channels are modulated by a variety of cellular lipids and pharmacological agents, including polyunsaturated fatty acids and volatile general anaesthetics. Recent in vivo studies have demonstrated that TREK1, the most thoroughly studied K(2P) channel, has a key role in the cellular mechanisms of neuroprotection, anaesthesia, pain and depression.

Second Bound State of PsH

The existence of a second bound state of PsH that is electronically stable and also stable against positron annihilation by the normal 2gamma and 3gamma processes is demonstrated by explicit calculation. The state can be found in the ;{2,4}S;{o} symmetries with the two electrons in a spin-triplet state. The binding energy against dissociation into the H(2p)+Ps(2p) channel was 7.03 x 10;(-4) hartree. The dominant decay mode of the states will be radiative decay into a configuration that autoionizes or undergoes positron annihilation. The NaPs system of the same symmetry is also electronically stable with a binding energy of 1.514 x 10;(-3) hartree with respect to the Na(3p)+Ps(2p) channel.

Chapter 7 - Regulation of the Mechano‐Gated K2P Channel TREK‐1 by Membrane Phospholipids

This chapter discusses the regulation of the mechano-gated K(2P) channel, TREK-1 by membrane phospholipids. TREK-1 (KCNK2 or K2P2.1) is a polymodal K(+) channel that is activated by membrane stretch, intracellular acidosis, heat, and cellular lipids, such as arachidonic acid (AA). Phospholipids, including PIP2, exert a dual dose-dependent effect on TREK-1. Low concentrations transform the mechanogated K(+) channel TREK-1 into a leak K(+) channel. The phospholipid-sensing domain is a positively charged cluster in the proximal C-terminal domain. This region also encompasses the proton sensor E306 that is required for the activation of TREK-1 by cytosolic acidosis. Protonation of E306 increases channel-phospholipid interaction leading to TREK-1 opening without direct-mechanical stimulation. At higher concentrations, intracellular phospholipids inhibit channel activation by stretch, intracellular acidosis, and AA. Binding endogenous negative inner leaflet phospholipids with polylysine reduces the inhibition and reveals channel stimulation by exogenous intracellular phospholipids. Both stimulatory and inhibitory effects are observed with phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidic acid (PA), but not diacylglycerol (DG), suggesting that the phosphate at position 3 is required, although the net charge is not critical. Membrane phospholipids, including PIP2, are major regulators of TREK-1 channel activity.

A novel acid-sensitive K + channel in rat dorsal root ganglia neurons

Recent studies have suggested that acid-sensitive background K + channels such as TASK-1 and TASK-3, members of two-pore domain K + (K 2P) channel family, express and contribute to extracellular acidification-induced responses in dorsal root ganglia (DRG) neurons. However, it has remained to address whether other acid-sensitive background K + channels are functionally expressed in DRG neurons. Here we characterized biophysical and pharmacological properties of a novel acid-sensitive background K + channel in DRG neurons isolated from neonatal rats. We recorded an 80-pS K + channel with a weak inward rectification current–voltage relationship in cell-attached patches in 150 mM KCl bath solution. The 80-pS K + channel was inhibited by extracellular low pH (pH o 6.3). Interestingly, the channel was similar to TASK-2 cloned from mouse and rat in biophysical and pharmacological properties. However, extracellular alkaline condition which activates TASK-2 channel, failed to activate the 80-pS K + channel. Lidocaine and quinine more inhibited the channel activity of 80-pS K + channel than that of TASK-2 channel. Our results suggest that the acid-sensitive 80-pS K + channels may regulate resting membrane potential and may play a critical role in various processes such as cell metabolism, pH, and pain sensation in DRG neurons.

Cross‐talk between the mechano‐gated K 2P channel TREK‐1 and the actin cytoskeleton

TREK-1 (KCNK2) is a K(2P) channel that is highly expressed in fetal neurons. This K(+) channel is opened by a variety of stimuli, including membrane stretch and cellular lipids. Here, we show that the expression of TREK-1 markedly alters the cytoskeletal network and induces the formation of actin- and ezrin-rich membrane protrusions. The genetic inactivation of TREK-1 significantly alters the growth cone morphology of cultured embryonic striatal neurons. Cytoskeleton remodelling is crucially dependent on the protein kinase A phosphorylation site S333 and the interactive proton sensor E306, but is independent of channel permeation. Conversely, the actin cytoskeleton tonically represses TREK-1 mechano-sensitivity. Thus, the dialogue between TREK-1 and the actin cytoskeleton might influence both synaptogenesis and neuronal electrogenesis.

The lipid‐activated two‐pore domain K+ channel TREK‐1 is resistant to hypoxia: implication for ischaemic neuroprotection

TREK-1 is a member of the two-pore domain potassium (K(2P)) channel family that is mechano-, heat, pH, voltage and lipid sensitive. It is highly expressed in the central nervous system and probably encodes one of the previously described arachidonic acid-activated K(+) channels. Polyunsaturated fatty acids and lysophospholipids protect the brain against global ischaemia. Since both lipids are openers of TREK-1, it has been suggested that this K(2P) channel is directly involved in neuroprotection. Recently, however, this view has been challenged by a report claiming that TREK-1 and its activation by arachidonic acid is inhibited by hypoxia. In the present study, we demonstrate that the bubbling of saline with gases results in the loss of arachidonic acid from solution. Using experimental conditions which obviate this experimental artefact we demonstrate that TREK-1 is resistant to hypoxia and is strongly activated by arachidonic acid even at low P(O(2)) (< 4 Torr). Furthermore, hypoxia fails to affect basal as well as 2,4,6-trinitrophenol- and acid-stimulated TREK-1 currents. These data are supportive for a possible role of TREK-1 in ischaemic neuroprotection and in cell signalling via arachidonic acid.

A phospholipid sensor controls mechanogating of the K+ channel TREK-1

TREK-1 (KCNK2 or K(2P)2.1) is a mechanosensitive K(2P) channel that is opened by membrane stretch as well as cell swelling. Here, we demonstrate that membrane phospholipids, including PIP(2), control channel gating and transform TREK-1 into a leak K(+) conductance. A carboxy-terminal positively charged cluster is the phospholipid-sensing domain that interacts with the plasma membrane. This region also encompasses the proton sensor E306 that is required for activation of TREK-1 by cytosolic acidosis. Protonation of E306 drastically tightens channel-phospholipid interaction and leads to TREK-1 opening at atmospheric pressure. The TREK-1-phospholipid interaction is critical for channel mechano-, pH(i)- and voltage-dependent gating.

Functional expression of TREK-2 in insulin-secreting MIN6 cells

Insulin secretion from pancreatic β cells is partly regulated by cell membrane potential. Background K + channels that stabilize the resting membrane potential would suppress excitability and insulin secretion. Recent studies show that members of the two-pore domain K + (K 2P) channel family behave as background K + channels in many excitable cells. Therefore, the expression of K 2P channels was studied in insulin-secreting MIN6 cells. Reverse transcriptase PCR showed that, among nine K 2P channels tested, TASK-1, TASK-2, TASK-3, TREK-2, and TRESK-2 were expressed in MIN6 cells. Cell-attached recordings on MIN6 cells revealed five types of K + channels that were open at rest. Two were ATP-sensitive and Ca 2+-activated K + channels, as judged by their sensitivity to ATP and Ca 2+, respectively, and single-channel conductance. Among five K 2P channels, only TREK-2 could be clearly identified in MIN6 cells. The molecular identity of two other K + channels is not yet known. TREK-2 in MIN6 cells was activated by arachidonic acid, membrane stretch, and low pH solution (pH 5.8). Arachidonic acid increased Ba 2+-sensitive whole-cell current in MIN6 cell. These results suggest that TREK-2 contributes to the background K + conductance in MIN6 cells, and may regulate depolarization-induced secretion of insulin.

Functional Expression of TRESK-2, a New Member of the Tandem-pore K+ Channel Family

A new member of the tandem-pore K+ (K(2P)) channel family has been isolated from mouse testis complementary DNA. The new K(2P) channel was named TRESK-2, as its amino acid sequence shares 65% identity with that of TRESK-1. Mouse TRESK-2 is a 394-amino acid protein and possesses four putative transmembrane segments and two pore-forming domains. TRESK-2 has a long cytoplasmic domain joining the second and third transmembrane segments and a short carboxyl terminus. In the rat, TRESK-2 mRNA transcripts were expressed abundantly in the thymus and spleen and at low levels in many other tissues, including heart, small intestine, skeletal muscle, uterus, testis, and placenta, as judged by Northern blot analysis. TRESK-2 mRNA was also expressed in mouse and human tissues. In COS-7 cells transfected with TRESK-2 DNA, a time-independent and noninactivating K+-selective current was recorded. TRESK-2 was insensitive to 1 mm tetraethylammonium, 100 nm apamin, 1 mm 4-aminopyridine, and 10 microm glybenclamide. TRESK-2 was inhibited by 10 microm quinidine, 20 microm arachidonate and acid (pH 6.3) at 49, 43, and 23%, respectively. Single channel openings of TRESK-2 showed marked open channel noise. In symmetrical 150 mm KCl, the current-voltage relationship of TRESK-2 was slightly inwardly rectifying, with the single channel conductance 13 picosiemens (pS) at +60 mV and 16 pS at -60 mV. In inside-out patches, TRESK-2 was unaffected by the intracellular application of 10 microm guanosine 5'-O-(thiotriphosphate). These results show that TRESK-2 is a functional member of the K(2P) channel family and contributes to the background K+ conductance in many types of cells.

Electron transfer from optically prepared states: He+ + Na(32P) → He(2 1,3P) + Na+ angular differential scattering

This paper reports experimental and theoretical scattering angle-dependent differential cross sections (DCS) for the electron transfer processes He+ + Na(3s or 3p) → He(2s or 2p) + Na+ at an impact energy of 1 keV. The He(2p) channels are the dominant contributors to electron transfer from the Na(3p) states. Beam experiments with optically prepared Na(3p) states are analysed using time-of-flight spectroscopy without resolution of the final He 21P and 23P states. The theoretical approach is based on a one-electron two-centre atomic orbital expansion of the electronic singlet and triplet scattering states, using the coupled-channel impact parameter method and the eikonal Bessel transformation to obtain quantal DCS predictions. The theoretical results show distinct differences between singlet and triplet channels. In spite of the smaller statistical weight, the singlet contribution about equals the triplet contribution due to the smaller singlet energy defect. For Na(3s) → He(2s) transfer DCS the theoretical results predict a Fraunhofer-type diffraction pattern as seen previously in Na(3s) → Li(2s) transfer, however, the rings are too narrow to be resolved in the present experiment. The spatial pattern of the scattered neutrals is strongly forwardly peaked with typical scattering angles of less than 0.05°. The results reveal pronounced spatial anisotropies, strongly dependent on the initial target preparation. In particular, when a Na(3p) orbital is prepared aligned along the direction of the initial ion beam, electron transfer is much more efficient than for the perpendicular orbital geometry. Furthermore, if the orbital is tilted by 45° with respect to the incident beam direction a strong left–right transfer scattering asymmetry is observed, with more neutrals being scattered to the side to which the p orbital is pointing than to the opposite side. The experimental observations compare well with the theoretical predictions for the spin-averaged electron transfer. Similarities and differences with earlier electron transfer studies for H+ and Li+ on Na(3p) at the same impact energy are pointed out.

Single-channel properties and pH sensitivity of two-pore domain K + channels of the TALK family

The two-pore K 2P channel family comprises TASK, TREK, TWIK, TRESK, TALK, and THIK subfamilies, and TALK-1, TALK-2, and TASK-2 are functional members of the TALK subfamily. Here we report for the first time the single-channel properties of TALK-2 and its pH o sensitivity, and compare them to those of TALK-1 and TASK-2. In transfected COS-7 cells, the three TALK K 2P channels could be identified easily by their differences in single-channel conductance and gating kinetics. The single-channel conductances of TALK-1, TALK-2, and TASK-2 in symmetrical 150 mM KCl were 21, 33, and 70 pS (−60 mV), respectively. TALK-2 was sensitive mainly to the alkaline range (pH 7–10), whereas TALK-1 and TASK-2 were sensitive to a wider pH o range (6–10). The effect of pH changes was mainly on the opening frequency. Thus, members of the TALK family expressed in native tissues may be identified based on their single-channel kinetics and pH o sensitivity.

Anesthetic-sensitive 2P domain K+ channels.

Anesthetic-sensitive 2P domain K+ channels.

Potential Energy Surfaces for LiH2 and Photochemical Reactions Li*+ H2 ↔ LiH + H

Potential energy surfaces for the Li*+ H2 → LiH + H and the reverse reactions are calculated using ab initio methods. Extensive configuration interactions have been done for a large number of collinear, C2v, and Cs geometrical forms using a large basis set to describe the 2s−3d atomic states of the lithium atom and the neutral and anionic hydrogen molecule. The Li(3s) channel has a small activation barrier and leads to a stable intermediate, 4 2A‘ (LiH2)*, but a diabatic coupling between the 3 and 4 2A‘ surfaces would preferentially lead to a nonreactive inelastic collision producing Li(2p). The Li(2p) channel leads to one entirely repulsive potential surface (3 2A‘) and two attractive potential surfaces (2 2A‘ and 1 2A‘ ‘) resulting in two stable intermediates. The Li(2p) atoms with enough collision energy to overcome the endothermicity of 1624 cm-1 can lead to the reaction producing LiH. Those three excited intermediate complexes are bent (planar) and not linear. The charge transfer from the metal atom to the hydrogen molecule does not occur in the course of the collision but much later, when one hydrogen atom breaks away, implying that the harpooning model does not apply to this case. The reverse reaction LiH + H → Li + H2 can produce (i) the high-energy Li(2s) directly, (ii) the thermal Li(2p), and (iii) the thermal Li(2s) accompanied with a chemiluminescence. The 2 2A‘ intermediate may play an important role in the reverse reaction, too.