Genomic and functional characteristics of novel human pancreatic 2P domain K(+) channels.

We isolated three novel 2P domain K(+) channel subunits from human. The first two subunits, TALK-1 and TALK-2, are distantly related to TASK-2. Their genes form a tight cluster of 25 kb on chromosome 6p21.1-p21.2. The corresponding channels produce quasi-instantaneous and non-inactivating currents that are activated at alkaline pHs. These currents are sensitive to Ba(2+), quinine, quinidine, chloroform, halothane, and isoflurane but are not affected by TEA, 4-AP, Cs(+), arachidonic acid, hypertonic solutions, agents activating protein kinases C and A, changes of internal Ca(2+) concentrations, and by activation of G(i) and G(q) proteins. TALK-1 is exclusively expressed in the pancreas. TALK-2 is mainly expressed in the pancreas, but is also expressed at a lower level in liver, placenta, heart, and lung. We also cloned a third subunit, named hTHIK-2 which is present in many tissues with high levels again in the pancreas but which could not be functionally expressed.

TWIK-2, an inactivating 2P domain K+ channel.

We cloned human and rat TWIK-2 and expressed this novel 2P domain K(+) channel in transiently transfected COS cells. TWIK-2 is highly expressed in the gastrointestinal tract, the vasculature, and the immune system. Rat TWIK-2 currents are about 15 times larger than human TWIK-2 currents, but both exhibit outward rectification in a physiological K(+) gradient and mild inward rectification in symmetrical K(+) conditions. TWIK-2 currents are inactivating at depolarized potentials, and the kinetic of inactivation is highly temperature-sensitive. TWIK-2 shows an extremely low conductance, which prevents the visualization of discrete single channel events. The inactivation and rectification are intrinsic properties of TWIK-2 channels. In a physiological K(+) gradient, TWIK-2 is half inhibited by 0.1 mm Ba(2+), quinine, and quinidine. Finally, cysteine 53 in the M1P1 external loop is required for functional expression of TWIK-2 but is not critical for subunit self-assembly. TWIK-2 is the first reported 2P domain K(+) channel that inactivates. The base-line, transient, and delayed activities of TWIK-2 suggest that this novel 2P domain K(+) channel may play an important functional role in cell electrogenesis.

Immunolocalization of the arachidonic acid and mechanosensitive baseline traak potassium channel in the nervous system.

TRAAK is the sole member of the emerging class of 2P domain K+ channels to be exclusively expressed in neuronal cells. TRAAK produces baseline K+ currents which are strongly stimulated by arachidonic acid and by mechanical stretch, and which are insensitive to the classical K+ channel blockers tetraethylammonium, Ba2+, and Cs+. This report describes the immunolocalization of TRAAK in brain, spinal cord, and retina of the adult mouse. The most striking finding is the widespread distribution of the TRAAK immunoreactivity, with a prominent staining of the cerebellar cortex, neocortex, hippocampus, dentate gyrus, subiculum, the dorsal hippocampal commissure, thalamus, caudate-putamen, olfactory bulb, and several nuclei in the brainstem. Virtually all neurons express TRAAK, and the highest immunoreactivity was seen in soma, and to a lesser degree in axons and/or dendrites in most areas in brain and spinal cord. In the retina, the TRAAK protein is concentrated to the soma of ganglion cells and to the dendrites of all other neurons. Taken together, these results show a wide distribution of TRAAK, a mechanosensitive and arachidonic acid-stimulated neuron-specific baseline K+ channel, in brain, spinal cord and retina.

Cloning of a new mouse two-P domain channel subunit and a human homologue with a unique pore structure.

Mouse KCNK6 is a new subunit belonging to the TWIK channel family. This 335-amino acid polypeptide has four transmembrane segments, two pore-forming domains, and a Ca2+-binding EF-hand motif. Expression of KCNK6 transcripts is principally observed in eyes, lung, stomach and embryo. In the eyes, immunohistochemistry reveals protein expression only in some of the retina neurons. Although KCNK6 is able to dimerize as other functional two-P domain K+ channels when it is expressed in COS-7 cells, it remains in the endoplasmic reticulum and is unable to generate ionic channel activity. Deletions, mutations, and chimera constructions suggest that KCNK6 is not an intracellular channel but rather a subunit that needs to associate with a partner, which remains to be discovered, in order to reach the plasma membrane. A closely related human KCNK7-A subunit has been cloned. KCNK7 displays an intriguing GLE sequence in its filter region instead of the G(Y/F/L)G sequence, which is considered to be the K+ channel signature. This subunit is alternatively spliced and gives rise to the shorter forms KCNK7-B and -C. None of the KCNK7 structures can generate channel activity by itself. The KCNK7 gene is situated on chromosome 11, in the q13 region, where several candidate diseases have been identified.

TRAAK is a mammalian neuronal mechano-gated K+ channel.

The novel structural class of mammalian channels with four transmembrane segments and two pore regions comprise background K+ channels (TWIK-1, TREK-1, TRAAK, TASK, and TASK-2) with unique physiological functions (1-6). Unlike its counterparts, TRAAK is only expressed in neuronal tissues, including brain, spinal cord, and retina (1). This report shows that TRAAK, which was known to be activated by arachidonic acid (3), is also opened by membrane stretch. Mechanical activation of TRAAK is induced by a convex curvature of the plasma membrane and can be mimicked by the amphipathic membrane crenator trinitrophenol. Cytoskeletal elements are negative tonic regulators of TRAAK. Membrane depolarization and membrane crenation synergize with stretch-induced channel opening. Finally, TRAAK is reversibly blocked by micromolar concentrations of gadolinium, a well known blocker of stretch-activated channels. Mechanical activation of TRAAK in the central nervous system may play an important role during growth cone motility and neurite elongation.

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.

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.

Coexistence of two classes of glibenclamide-inhibitable ATP-regulated K+ channels in avian skeletal muscle.

Avian skeletal muscle expresses two types of ATP-sensitive K+ channels which have a unitary conductance of 15pS. These K+ channels can be distinguished pharmacologically by their high or low sensitivity to the antidiabetic sulphonylurea blocker glibenclamide. Both channels are activated by the K+ channel opener cromakalim. Chick skeletal muscle expresses high-affinity binding sites for [3H]glibenclamide (Kd = 0.6nM) which presumably correspond to the ATP-sensitive K+ channels with the greatest sensitivity to glibenclamide. The density of these high-affinity binding sites varies during muscle development. The maximum density (500fmol/mg protein) appears at 16 days in ovo, i.e. at a period when myoblasts have differentiated into myotubes and when innervation of myotubes has started. After this maximum, the level of [3H]glibenclamide-binding sites decreases to a plateau value of 100fmol/mg protein at 2-5 days post-natal. When muscle cells are put in cultures, the high-affinity binding sites disappear rapidly. Neither glibenclamide nor cromakalim have any effect on normal physiological chick muscle contraction. They have no effect on contracture and/or 86Rb+ efflux produced by metabolic poisoning.

Receptor binding characterization in kidney membrane of [3H]U-37883, a novel ATP-sensitive K+ channel blocker with diuretic/natriuretic properties.

U-37883 (4-morpholinecarboximidine-N-1-adamantyl-N-cyclohexyl), a known blocker of ATP-sensitive K+ (KATP) channels, produces natriuresis/diuresis in vivo by a direct effect on the kidney. In the present study, the binding characteristics of the U-37883 receptor were investigated using pig kidney cortex microsomes. [3H]U-37883 (0.5-5 nM, 50 Ci/mmol) exhibited specific binding, which was reversible, increased linearly with protein concentration (50-500 micrograms/ml), and was destroyed after treatment with proteases. Scatchard plots derived from the competition experiments suggested the presence of a single class of low affinity binding sites, with a Kd of 225 nM and a Bmax of 7.8 pmol/mg of protein. A similar Kd value was derived from complementary studies dealing with association and dissociation kinetics. The binding of [3H]U-37883 was tissue specific, because very little specific binding could be detected in microsomes from rat insulinoma cells (RINm5F) and brain. In contrast, these membranes displayed high affinity specific binding of [3H]glyburide, another KATP channel blocker. Finally, analogs of U-37883 that were found to be active KATP channel blockers in isolated rabbit mesenteric artery and active in vivo as diuretics/natriuretics were also found to be active in displacing specific binding of [3H]U-37883, whereas the inactive analogs (no vascular KATP channel-blocking activity and no in vivo diuresis/natriuresis) were inactive in this binding assay. We suggest that the U-37883 binding site represents a functional receptor that mediates the KATP channel antagonism and natriuresis observed with this class of compounds.

Functional receptors in Xenopus oocytes for U-37883A, a novel ATP-sensitive K+ channel blocker: comparison with rat insulinoma cells.

Follicle-enclosed Xenopus oocytes were used to describe the ATP-sensitive K+ (KATP) channel-blocking properties of U-37883A (4-morpholinecarboximidine-N-1-adamantyl-N'-cyclohexyl), in comparison with glibenclamide. In follicular oocytes, the KATP channel opener P1060 (30 microM), a pinacidil analog, activated a large outward K+ current that was blocked by glibenclamide (IC50 = 0.33 microM) and U-37883A (IC50 = 0.26 microM). P1060 activation was inhibited by both U-37883A and glibenclamide in a noncompetitive manner. U-37883A also blocked the KATP channel activation by cAMP (300 microM) and adenosine (10 microM). Single-channel studies on isolated follicular cells showed that U-37883A (10 microM) reduced the open probability of the KATP channel by 76%, without significantly modifying the single-channel current amplitude. Receptor binding studies with [3H]U-37883 in membranes from follicle-enclosed oocytes demonstrated a single class of low affinity binding sites, with a Kd of 450 nM and a Bmax of 17 pmol/mg of protein. Studies with analogs of U-37883A showed that U-52090A inhibited KATP current and displaced [3H]U-37883 from its binding site with similar potencies. In contrast, U-42069D neither inhibited KATP current nor competed with [3H]U-37883 binding. In RINm5F cells (an insulinoma cell line), U-37883A, unlike glibenclamide, failed to inhibit KATP current. Furthermore, there was no significant specific binding of [3H]U-37883 in RINm5F cell membranes, which displayed high levels of specific binding of [3H]glibenclamide. These data demonstrate the presence of a receptor for U-37883A-type guanidines that controls the activity of the endogenous KATP channels in follicle-enclosed oocytes. The available data collectively suggest that U-37883A is a more selective blocker of the follicular KATP channel, which is very similar to that in smooth muscle, than of the pancreatic beta cell KATP channel.

Immunohistochemical localization of L-type calcium channels in the developing first molar of the rat during odontoblast differentiation.

To study the presence of L-type calcium channels during the different steps of odontoblast differentiation, a specific monoclonal antibody against 1,4-dihydropyridine receptors was used in combination with avidin-biotin-peroxidase complex labelling. Staining was seen on the cell bodies of pre-odontoblasts, concentrated at the apical pole (distal portion) of functional odontoblasts and localized on cell bodies and processes of mature odontoblasts. Thus calcium channels were expressed at the onset of differentiation and maintained in the differentiated cells but with some changes of localization. It is suggested that these channels may facilitate the entry of calcium to act as a second messenger for cellular polarization or be involved in the transcellular transport of calcium to the mineralizing front.

ATP-modulated K+ channels sensitive to antidiabetic sulfonylureas are present in adenohypophysis and are involved in growth hormone release.

The adenohypophysis contains high-affinity binding sites for antidiabetic sulfonylureas that are specific blockers of ATP-sensitive K+ channels. The binding protein has a M(r) of 145,000 +/- 5000. The presence of ATP-sensitive K+ channels (26 pS) has been demonstrated by electrophysiological techniques. Intracellular perfusion of adenohypophysis cells with an ATP-free medium to activate ATP-sensitive K+ channels induces a large hyperpolarization (approximately 30 mV) that is antagonized by antidiabetic sulfonylureas. Diazoxide opens ATP-sensitive K+ channels in adenohypophysis cells as it does in pancreatic beta cells and also induces a hyperpolarization (approximately 30 mV) that is also suppressed by antidiabetic sulfonylureas. As in pancreatic beta cells, glucose and antidiabetic sulfonylureas depolarize the adenohypophysis cells and thereby indirectly increase Ca2+ influx through L-type Ca2+ channels. The K+ channel opener diazoxide has an opposite effect. Opening ATP-sensitive K+ channels inhibits growth hormone secretion and this inhibition is eliminated by antidiabetic sulfonylureas.

Effectors of ATP-sensitive K+ channels inhibit the regulatory effects of somatostatin and GH-releasing factor on growth hormone secretion.

Somatostatin inhibition of growth hormone (GH) secretion from adenohypophysis cells in culture was antagonized by the antidiabetic sulfonylurea glipizide (K0.5 = 10 +/- 5 nM). Although all cells that hyperpolarize with somatostatin have ATP-sensitive K+ channels, the antagonistic actions of the hormone and of the antidiabetic drug are due to effects on different types of K+ channels. Diazoxide, an opener of ATP-sensitive K+ channels, abolished the increase of intracellular Ca2+ provoked by growth hormone releasing factor (GRF) and induced inhibition of GRF stimulated GH secretion (K0.5 = 138 microM). This inhibition by diazoxide was largely suppressed by glipizide which blocked the ATP-sensitive K+ channels opened by diazoxide. In summary, hormonal activation of GH secretion is inhibited by openers of ATP-sensitive K+ channels, while hormonal inhibition of GH secretion is suppressed by blockers of ATP-sensitive K+ channels.

Antidiabetic sulfonylureas antagonize somatostatin inhibition of prolactin secretion in vitro.

Somatostatin-induced inhibition of prolactin secretion from adenohypophysis cells in culture is antagonized by the sulfonylurea glipizide, a specific blocker of ATP-sensitive K+ channels. The affinity constant for glipizide is K0.5 = 10 +/- 5 nM.

ATP/ADP binding sites are present in the sulfonylurea binding protein associated with brain ATP-sensitive K+ channels.

Covalent labeling of nucleotide binding sites of the purified sulfonylurea receptor has been carried out with alpha-32P-labeled oxidized ATP. The main part of 32P incorporation is in the 145-kDa glycoprotein that has been previously shown to be the sulfonylurea binding protein (Bernardi et al., 1988). ATP and ADP protect against this covalent labeling with K0.5 values of 100 microM and 500 microM, respectively. Non-hydrolyzable analogs of ATP also inhibit 32P incorporation. Interactions between nucleotide binding sites and sulfonylurea binding sites have then been observed. AMP-PNP, a nonhydrolyzable analog of ATP, produces a small inhibition of [3H]glibenclamide binding (20-25%) which was not influenced by Mg2+. Conversely, ADP, which also produced a small inhibition (20%) in the absence of Mg2+, produced a large inhibition (approximately 80%) in the presence of Mg2+. This inhibitory effect of the ADP-Mg2+ complex was observed with a K0.5 value of 100 +/- 40 microM. All the results taken together indicate that ATP and ADP-Mg2+ binding sites that control the activity of KATP channels are both present on the same subunit that bears the receptors for antidiabetic sulfonylureas.

K+ channel openers activate brain sulfonylurea-sensitive K+ channels and block neurosecretion.

Vascular K+ channel openers such as cromakalim, nicorandil, and pinacidil potently stimulate 86Rb+ efflux from slices of substantia nigra. This 86Rb+ efflux is blocked by antidiabetic sulfonylureas, which are known to be potent and specific blockers of ATP-regulated K+ channels in pancreatic beta cells, cardiac cells, and smooth muscle cells. K0.5, the half-maximal effect of the enantiomer (-)-cromakalim, is as low as 10 nM, whereas K0.5 for nicorandil is 100 nM. These two compounds appear to have a much higher affinity for nerve cells than for smooth muscle cells. Openers of sulfonylurea-sensitive K+ channels lead to inhibition of gamma-aminobutyric acid release. There is an excellent relationship between potency to activate 86Rb+ efflux and potency to inhibit neurotransmitter release.

Glucose, sulfonylureas, and neurotransmitter release: role of ATP-sensitive K+ channels.

Sulfonylurea-sensitive adenosine triphosphate (ATP)-regulated potassium (KATP) channels are present in brain cells and play a role in neurosecretion at nerve terminals. KATP channels in substantia nigra, a brain region that shows high sulfonylurea binding, are inactivated by high glucose concentrations and by antidiabetic sulfonylureas and are activated by ATP depletion and anoxia. KATP channel inhibition leads to activation of gamma-aminobutyric acid (GABA) release, whereas KATP channel activation leads to inhibition of GABA release. These channels may be involved in the response of the brain to hyper- and hypoglycemia (in diabetes) and ischemia or anoxia.