Human TASK-1 Research Articles

Determinants of the Anesthetic Sensitivity of Two-pore Domain Acid-sensitive Potassium Channels: MOLECULAR CLONING OF AN ANESTHETIC-ACTIVATED POTASSIUM CHANNEL FROM LYMNAEA STAGNALIS

Certain two-pore domain K(+) channels are plausible targets for volatile general anesthetics, yet little is known at the molecular level about how these simple agents cause channel activation. The first anesthetic-activated K(+) current I(K(An)) that was characterized was discovered in the mollusk Lymnaea stagnalis and is remarkable for both its sensitivity to general anesthetics and its stereoselective responses to anesthetic enantiomers (Franks, N. P., and Lieb, W. R. (1988) Nature 333, 662-664 and Franks, N. P., and Lieb, W. R. (1991) Science 254, 427-430). Here we report the molecular cloning of a two-pore domain K(+) channel LyTASK from L. stagnalis and show that, when expressed in HEK-293 cells, it displays the same biophysical characteristics as the anesthetic-activated K(+) current I(K(An)). Sequence analysis and functional properties show it to be a member of the TASK family of channels with approximately 47% identity at the amino acid level when compared with human TASK-1 and TASK-3. By using chimeric channel constructs and site-directed mutagenesis we have identified the specific amino acid 159 to be a critical determinant of anesthetic sensitivity, which, when mutated to alanine, essentially eliminates anesthetic activation in the human channels and greatly reduces activation in LyTASK. The L159A mutation in LyTASK disrupts the stereoselective response to isoflurane while having no effect on the pH sensitivity of the channel, suggesting this critical amino acid may form part of an anesthetic binding site.

A Di‐Acidic Sequence Motif Enhances the Surface Expression of the Potassium Channel TASK‐3

We have characterized a sequence motif, EDE, in the proximal C-terminus of the acid-sensitive potassium channel TASK-3. Human TASK-3 channels were expressed in Xenopus oocytes, and the density of the channels at the surface membrane was studied with two complementary techniques: a luminometric surface expression assay of hemagglutinin epitope-tagged TASK-3 channels and voltage-clamp measurements of the acid-sensitive potassium current. Both approaches showed that mutation of the two glutamate residues of the EDE motif to alanine (ADA mutant) markedly reduced the transport of TASK-3 channels to the cell surface. Mutation of the central aspartate of the EDE motif had no effect on surface expression. The functional role of the EDE motif was further characterized in chimaeric constructs consisting of truncated Kir2.1 channels to which the C-terminus of TASK-3 was attached. In these constructs, too, replacement of the EDE motif by ADA strongly reduced surface expression. Live-cell imaging of enhanced green fluorescent protein-tagged channels expressed in COS-7 cells showed that 24 h after transfection wild-type TASK-3 was mainly localized to the cell surface whereas the ADA mutant was largely retained in the endoplasmic reticulum (ER). Mutation of a second di-acidic motif in the C-terminus of TASK-3 (DAE) had no effect on surface expression. Coexpression of TASK-3 with a GTP-restricted mutant of the coat recruitment GTPase Sar1 (Sar1H79G) resulted in ER retention of the channel. Our data suggest that the di-acidic motif, EDE, in human TASK-3 is a major determinant of the rate of ER export and is required for efficient surface expression of the channel.

Expression of a two‐pore domain K+ channel (TASK‐1) in developing avian and mouse ventricular conduction systems

In this study, we report the identification and amino acid sequence of a novel two-pore domain potassium channel (TASK-1) in chicken. This protein, cTASK-1, is highly similar to mouse and human TASK-1 particularly within the pore regions. We describe the expression profile of both chicken and mouse TASK-1 in the embryonic heart as the ventricular conduction system develops. The developmental distribution of TASK-1 is similar in chicken and mouse. Initially, TASK-1 is expressed throughout the myocardium of the early heart tube. However, as cardiogenesis proceeds, ventricular expression becomes restricted to the trabeculated myocardium and eventually the bundle of His, bundle branches, and Purkinje fibers of the mature conduction system. This finding suggests that components of the ventricular conduction system differentiate from TASK-1-positive myocytes of the early heart tube that retain TASK-1 expression as they mature. Our results are consistent with a common mechanism for ventricular conduction system development in avians and mammals, despite differences in the anatomy of the mature conduction systems of these organisms.

Selective block of the human 2-P domain potassium channel, TASK-3, and the native leak potassium current, IKSO, by zinc.

Background potassium channels control the resting membrane potential of neurones and regulate their excitability. Two-pore-domain potassium (2-PK) channels have been shown to underlie a number of such neuronal background currents. Currents through human TASK-1, TASK-2 and TASK-3 channels expressed in Xenopus oocytes were inhibited by extracellular acidification. For TASK-3, mutation of histidine 98 to aspartate or alanine considerably reduced this effect of pH. Zinc was found to be a selective blocker of TASK-3 with virtually no effect on TASK-1 or TASK-2. Zinc had an IC(50) of 19.8 microM for TASK-3, at +80 mV, with little voltage dependence associated with this inhibition. TASK-3 H98A had a much reduced sensitivity to zinc suggesting this site is important for zinc block. Surprisingly, TASK-1 also has histidine in position 98 but is insensitive to zinc block. TASK-3 and TASK-1 differ at position 70 with glutamate for TASK-3 and lysine for TASK-1. TASK-3 E70K also had a much reduced sensitivity to zinc while the corresponding reverse mutation in TASK-1, K70E, induced zinc sensitivity. A TASK-3-TASK-1 concatamer channel was comparatively zinc insensitive. For TASK-3, it is concluded that positions E70 and H98 are both critical for zinc block. The native cerebellar granule neurone (CGN) leak current, IK(SO), is sensitive to block by zinc, with current reduced to 0.58 of control values in the presence of 100 microM zinc. This suggests that TASK-3 channels underlie a major component of IK(SO). It has recently been suggested that zinc is released from inhibitory synapses onto CGNs. Therefore it is possible that inhibition of IK(SO) in cerebellar granule cells by synaptically released zinc may have important physiological consequences.

Anesthetic-sensitive 2P domain K+ channels.

Anesthetic-sensitive 2P domain K+ channels.

Functional characterisation of human TASK-3, an acid-sensitive two-pore domain potassium channel

Human TASK-3 (hTASK-3) is a recently identified member of the two-pore domain potassium channel (2PDKC) family which in man is predominantly expressed in the cerebellum. Previous preliminary examination of this channel indicates that when expressed in Xenopus oocytes, it produces a K + selective background conductance and consequent shift in resting membrane potential, thus mimicking other 2PDKC. Here we describe some additional functional and pharmacological aspects of hTASK-3-mediated conductances expressed in both Xenopus oocytes and HEK293 cells. hTASK-3 expression produces steady-state currents that approximate Goldman–Hodgkin–Katz behaviour with respect to membrane potential. Despite this, voltage steps from −80 mV to potentials >∼−20 mV induce currents that exhibit a clear time-dependent increase in current amplitude. Kinetically, this increase in current was well fit by a single exponential, the time constant of which was ∼10 ms and appeared independent of test potential, between −20 and +80 mV. In HEK293 cells hTASK-3 currents were inhibited by extracellular acidosis with a mid-point for inhibition of pH 6.4. Furthermore, the activity of TASK-3 was potentiated by the volatile anaesthetic halothane but inhibited by the local anaesthetic bupivacaine.

Cloning, localisation and functional expression of a novel human, cerebellum specific, two pore domain potassium channel

We have isolated, by degenerate PCR, a complementary DNA encoding a novel two pore domain potassium channel. This is the 7th functional member of the human tandem pore domain potassium channel family to be reported. It has an open reading frame of 1.125 kb and encodes a 374 amino acid protein which shows 62% identity to the human TASK-1 gene: identity to other human members of the family is 31–35% at the amino acid level. We believe this gene to be human TASK-3, the orthologue of the recently reported rat TASK-3 gene: amino acid identity between the two is 74%. ‘Taqman’ mRNA analysis demonstrated a very specific tissue distribution pattern, showing human TASK-3 mRNA to be localised largely in the cerebellum, in contrast rat TASK-3 was reported to be widely distributed. We have shown by radiation hybrid mapping that human TASK-3 can be assigned to chromosome 8q24.3. Human TASK-3 was demonstrated to endow Xenopus oocytes with a negative resting membrane potential through the presence of a large K + selective conductance. TASK-3 is inhibited by extracellular acidosis with a mid-point of inhibition around pH 6.5, supporting the predictions from the sequence data that this is a third human TASK (TWIK-related acid sensitive K + channel) gene.