Michel Fink

TASK, a human background K+ channel to sense external pH variations near physiological pH.

TASK is a new member of the recently recognized TWIK K+ channel family. This 395 amino acid polypeptide has four transmembrane segments and two P domains. In adult human, TASK transcripts are found in pancreas

Inhalational anesthetics activate two-pore-domain background K + channels

Volatile anesthetics produce safe, reversible unconsciousness, amnesia and analgesia via hyperpolarization of mammalian neurons. In molluscan pacemaker neurons, they activate an inhibitory synaptic K+ current (IKAn), proposed to be important in general anesthesia. Here we show that TASK and TREK-1, two recently cloned mammalian two-P-domain K+ channels similar to IKAn in biophysical properties, are activated by volatile general anesthetics. Chloroform, diethyl ether, halothane and isoflurane activated TREK-1, whereas only halothane and isoflurane activated TASK. Carboxy (C)-terminal regions were critical for anesthetic activation in both channels. Thus both TREK-1 and TASK are possibly important target sites for these agents.

A mammalian two pore domain mechano‐gated S‐like K+ channel

Aplysia S‐type K+ channels of sensory neurons play a dominant role in presynaptic facilitation and behavioural sensitization. They are closed by serotonin via cAMP‐dependent phosphorylation, whereas they are opened by arachidonic acid, volatile general anaesthetics and mechanical stimulation. We have identified a cloned mammalian two P domain K+ channel sharing the properties of the S channel. In addition, the recombinant channel is opened by lipid bilayer amphipathic crenators, while it is closed by cup‐formers. The cytoplasmic C‐terminus contains a charged region critical for chemical and mechanical activation, as well as a phosphorylation site required for cAMP inhibition.

TWIK-1, a ubiquitous human weakly inward rectifying K+ channel with a novel structure.

A new human weakly inward rectifying K+ channel, TWIK‐1, has been isolated. This channel is 336 amino acids long and has four transmembrane domains. Unlike other mammalian K+ channels, it contains two pore‐forming regions called P domains. Genes encoding structural homologues are present in the genome of Caenorhabditis elegans. TWIK‐1 currents expressed in Xenopus oocytes are time‐independent and present a nearly linear I‐V relationship that saturated for depolarizations positive to O mV in the presence of internal Mg2+. This inward rectification is abolished in the absence of internal Mg2+. TWIK‐1 has a unitary conductance of 34 pS and a kinetic behaviour that is dependent on the membrane potential. In the presence of internal Mg2+, the mean open times are 0.3 and 1.9 ms at −80 and +80 mV, respectively. The channel activity is up‐regulated by activation of protein kinase C and down‐regulated by internal acidification. Both types of regulation are indirect. TWIK‐1 channel activity is blocked by Ba2+(IC50=100 microM), quinine (IC50=50 microM) and quinidine (IC50=95 microM). This channel is of particular interest because its mRNA is widely distributed in human tissues, and is particularly abundant in brain and heart. TWIK‐1 channels are probably involved in the control of background K+ membrane conductances.

Cloning, functional expression and brain localization of a novel unconventional outward rectifier K+ channel.

Human TWIK‐1, which has been cloned recently, is a new structural type of weak inward rectifier K+ channel. Here we report the structural and functional properties of TREK‐1, a mammalian TWIK‐1‐related K+ channel. Despite a low amino acid identity between TWIK‐1 and TREK‐1 (approximately 28%), both channel proteins share the same overall structural arrangement consisting of two pore‐forming domains and four transmembrane segments (TMS). This structural similarity does not give rise to a functional analogy. K+ currents generated by TWIK‐1 are inwardly rectifying while K+ currents generated by TREK‐1 are outwardly rectifying. These channels have a conductance of 14 pS. TREK‐1 currents are insensitive to pharmacological agents that block TWIK‐1 activity such as quinine and quinidine. Extensive inhibitions of TREK‐1 activity are observed after activation of protein kinases A and C. TREK‐1 currents are sensitive to extracellular K+ and Na+. TREK‐1 mRNA is expressed in most tissues and is particularly abundant in the lung and in the brain. Its localization in this latter tissue has been studied by in situ hybridization. TREK‐1 expression is high in the olfactory bulb, hippocampus and cerebellum. These results provide the first evidence for the existence of a K+ channel family with four TMS and two pore domains in the nervous system of mammals. They also show that different members in this structural family can have totally different functional properties.

A neuronal two P domain K channel stimulated by arachidonic acid and polyunsaturated fatty acids

TWIK‐1, TREK‐1 and TASK K+ channels comprise a class of pore‐forming subunits with four membrane‐spanning segments and two P domains. Here we report the cloning of TRAAK, a 398 amino acid protein which is a new member of this mammalian class of K+ channels. Unlike TWIK‐1, TREK‐1 and TASK which are widely distributed in many different mouse tissues, TRAAK is present exclusively in brain, spinal cord and retina. Expression of TRAAK in Xenopus oocytes and COS cells induces instantaneous and non‐inactivating currents that are not gated by voltage. These currents are only partially inhibited by Ba2+ at high concentrations and are insensitive to the other classical K+ channel blockers tetraethylammonium, 4‐aminopyridine and Cs+. A particularly salient feature of TRAAK is that they can be stimulated by arachidonic acid (AA) and other unsaturated fatty acids but not by saturated fatty acids. These channels probably correspond to the functional class of fatty acid‐stimulated K+ currents that recently were identified in native neuronal cells but have not yet been cloned. These TRAAK channels might be essential in normal physiological processes in which AA is known to play an important role, such as synaptic transmission, and also in pathophysiological processes such as brain ischemia. TRAAK channels are stimulated by the neuroprotective drug riluzole.

Cloning and Expression of a Novel pH-sensitive Two Pore Domain K+ Channel from Human Kidney

A complementary DNA encoding a novel K+ channel, called TASK-2, was isolated from human kidney and its gene was mapped to chromosome 6p21. TASK-2 has a low sequence similarity to other two pore domain K+ channels, such as TWIK-1, TREK-1, TASK-1, and TRAAK (18–22% of amino acid identity), but a similar topology consisting of four potential membrane-spanning domains. In transfected cells, TASK-2 produces noninactivating, outwardly rectifying K+ currents with activation potential thresholds that closely follow the K+equilibrium potential. As for the related TASK-1 and TRAAK channels, the outward rectification is lost at high external K+concentration. The conductance of TASK-2 was estimated to be 14.5 picosiemens in physiological conditions and 59.9 picosiemens in symmetrical conditions with 155 mm K+. TASK-2 currents are blocked by quinine (IC50 = 22 μm) and quinidine (65% of inhibition at 100 μm) but not by the other classical K+ channel blockers tetraethylammonium, 4-aminopyridine, and Cs+. They are only slightly sensitive to Ba2+, with less than 17% of inhibition at 1 mm. As TASK-1, TASK-2 is highly sensitive to external pH in the physiological range. 10% of the maximum current was recorded at pH 6.5 and 90% at pH 8.8. Unlike all other cloned channels with two pore-forming domains, TASK-2 is essentially absent in the brain. In human and mouse, TASK-2 is mainly expressed in the kidney, where in situ hybridization shows that it is localized in cortical distal tubules and collecting ducts. This localization, as well as its functional properties, suggest that TASK-2 could play an important role in renal K+ transport.

Cloning provides evidence for a family of inward rectifier and G-protein coupled K+ channels in the brain

MbIRK3, mbGIRK2 and mbGIRK3 K+ channels cDNAs have been cloned from adult mouse brain. These eDNAs encode polypeptides of 445, 414 and 376 amino acids, respectively, which display the hallmarks of inward rectifier K+ channels, i.e. two hydrophobic membrane‐spanning domains M1 and M2 and a pore‐forming domain H5. MbIRK3 shows around 65% amino acid identity with IRK1 and rbIRK2 and only 50% with ROMK1 and GIRK1. On the other hand, mbGIRK2 and mbGIRK3 are more similar to GIRK1 (60%) than to ROMK1 and IRK1 (50%). Northern blot analysis reveals that these three novel clones are mainly expressed in the brain. Xenopus oocytes injected with mbIRK3 and mbGIRK2 cRNAs display inward rectifier K+‐selective currents very similar to IRK1 and GIRK1, respectively. As expected from the sequence homology, mbGIRK2 cRNA directs the expression of G‐protein coupled inward rectifyer K+ channels which has been observed through their functional coupling with co‐expressed δ‐opioid receptors. These results provide the first evidence that the GIRK family, as the IRK family, is composed of multiple genes with members specifically expressed in the nervous system.

Molecular Properties of Neuronal G-protein-activated Inwardly Rectifying K+ Channels

Four cDNA-encoding G-activated inwardly rectifying K+ channels have been cloned recently (Kubo, Y., Reuveny, E., Slesinger, P. A., Jan, Y. N., and Jan, L. Y.(1993) Nature 364, 802-806; Lesage, F., Duprat, F., Fink, M., Guillemare, E., Coppola, T., Lazdunski, M., and Hugnot, J. P. (1994) FEBS Lett. 353, 37-42; Krapivinsky, G., Gordon, E. A., Wickman, K., Velimirovic, B., Krapivinsky, L., and Clapham, D. E. (1995) Nature 374, 135-141). We report the cloning of a mouse GIRK2 splice variant, noted mGIRK2A. Both channel proteins are functionally expressed in Xenopus oocytes upon injection of their cRNA, alone or in combination with the GIRK1 cRNA. Three GIRK channels, mGIRK1-3, are shown to be present in the brain. Colocalization in the same neurons of mGIRK1 and mGIRK2 supports the hypothesis that native channels are made by an heteromeric subunit assembly. GIRK3 channels have not been expressed successfully, even in the presence of the other types of subunits. However, GIRK3 chimeras with the amino- and carboxyl-terminal of GIRK2 are functionally expressed in the presence of GIRK1. The expressed mGIRK2 and mGIRK1, −2 currents are blocked by Ba2+ and Cs+ ions. They are not regulated by protein kinase A and protein kinase C. Channel activity runs down in inside-out excised patches, and ATP is required to prevent this rundown. Since the nonhydrolyzable ATP analog AMP-PCP is also active and since addition of kinases A and C as well as alkaline phosphatase does not modify the ATP effect, it is concluded that ATP hydrolysis is not required. An ATP binding process appears to be essential for maintaining a functional state of the neuronal inward rectifier K+ channel. A Na+ binding site on the cytoplasmic face of the membrane acts in synergy with the ATP binding site to stabilize channel activity.

Dimerization of TWIK-1 K+ channel subunits via a disulfide bridge.

TWIK‐1 is a new type of K+ channel with two P domains and is abundantly expressed in human heart and brain. Here we show that TWIK‐1 subunits can self‐associate to give dimers containing an interchain disulfide bridge. This assembly involves a 34 amino acid domain that is localized to the extracellular M1P1 linker loop. Cysteine 69 which is part of this interacting domain is implicated in the formation of the disulfide bond. Replacing this cysteine with a serine residue results in the loss of functional K+ channel expression. This is the first example of a covalent association of functional subunits in voltage‐sensitive channels via a disulfide bridge.