Alteration and restoration of K+ channel function by deletions at the N- and C-termini

Voltage-dependent ion channels are thought to consist of a highly conserved repeated core of six transmembrane segments, flanked by more variable cytoplasmic domains. Significant functional differences exist among related types of K+ channels. These differences have been attributed to the variable domains, most prominently the N- and C-termini. We have therefore investigated the functional importance of both termini for the delayed rectifier K+ channel from rat brain encoded by the drk1 gene. This channel has an unusually long C-terminus. Deletions in either terminus affected both activation and inactivation, in some cases profoundly. Unexpectedly, more extensive deletions in both termini restored gating. We could therefore define a core region only slightly longer than the six transmembrane segments that is sufficient for the formation of channels with the kinetics of a delayed rectifier.     

Cloning and expression of a human voltage-gated potassium channel. A novel member of the RCK potassium channel family.

We have isolated and characterized a human cDNA (HBK2) that is homologous to novel member (RCK2) of the K+ channel RCK gene family expressed in rat brain. RCK2 mRNA was detected predominantly in midbrain areas and brainstem. The primary sequences of the HBK2/RCK2 K+ channel proteins exhibit major differences to other members of the RCK gene family. The bend region between segments S1 and S2 is unusually long and does not contain the N-glycosylation site commonly found in this region. They might be O-glycosylated instead. Functional characterization of the HBK2/RCK2 K+ channels in Xenopus laevis oocytes following micro-injection in in vitro transcribed HBK2 or RCK2 cRNA showed that the HBK2/RCK2 proteins form voltage-gated K+ channels with novel functional and pharmacological properties. These channels are different to RCK1, RCK3, RCK4 and RCK5 K+ channels.     

At least two mRNA species contribute to the properties of rat brain A-type potassium channels expressed in Xenopus oocytes

Fast transient K+ channels (A channels) of the type operating in the subthreshold region for Na+ action potential generation were expressed in Xenopus oocytes injected with rat brain poly(A) RNA. Sucrose gradient fractionation of the RNA separates mRNAs encoding A-currents (6-7 kb) from mRNAs encoding other voltage-dependent K+ channels. A-currents expressed with fractionated mRNA differ in kinetics and pharmacology from A-currents expressed with total mRNA. The original properties of the A-currents can be reconstituted when small mRNAs (2-4 kb) are added to the large mRNA fraction. Thus the properties of the A-currents expressed with total poly(A) RNA depend on the presence of more than one mRNA species. mRNA(s) present in the large RNA fraction must encode channel subunits since they express an A-current by themselves. The small mRNA(s) may encode a second subunit(s) or a factor, such as an enzymatic activity that modulates the properties of the channels, which could play a role in generating A-channel functional diversity.     

Localization and molecular determinants of the Hanatoxin receptors on the voltage-sensing domains of a K(+) channel

Hanatoxin inhibits voltage-gated K(+) channels by modifying the energetics of activation. We studied the molecular determinants and physical location of the Hanatoxin receptors on the drk1 voltage-gated K(+) channel. First, we made multiple substitutions at three previously identified positions in the COOH terminus of S3 to examine whether these residues interact intimately with the toxin. We also examined a region encompassing S1-S3 using alanine-scanning mutagenesis to identify additional determinants of the toxin receptors. Finally, guided by the structure of the KcsA K(+) channel, we explored whether the toxin interacts with the peripheral extracellular surface of the pore domain in the drk1 K(+) channel. Our results argue for an intimate interaction between the toxin and the COOH terminus of S3 and suggest that the Hanatoxin receptors are confined within the voltage-sensing domains of the channel, at least 20-25 A away from the central pore axis.     

Characterization of a mammalian cDNA for an inactivating voltage-sensitive K+ channel.

A cDNA clone encoding a K+ channel polypeptide with 72% amino acid sequence identity to Drosophila Shal was isolated from rat hippocampus. Functional expression of the cDNA in Xenopus oocytes generated 4-amino-pyridine-sensitive K+ channels displaying rapid inactivation kinetics. The fastest component of inactivation was slowed by the deletion of 3 basic residues in the amino-terminal region. Northern blots revealed that the mRNA encoding this K+ channel polypeptide was expressed at a similar level in the brain and in the heart. In situ hybridization revealed that the mRNA encoding this K+ channel appeared concentrated in the hippocampus, dentate gyrus, and habenular nucleus in the brain. Thus, this K+ channel polypeptide is likely to form some of the A-type K+ channels expressed in the mammalian nervous system and heart.     

Cloning and functional expression of a TEA-sensitive A-type potassium channel from rat brain.

A rat brain cDNA (Raw3) related to the Drosophila Shaw K+ channel family has been characterized. Raw3 cRNA leads to the formation of TEA-insensitive, fast inactivating (A-type) K+ channels when injected into Xenopus laevis oocytes. Raw3 channels have markedly different properties from the previously cloned rat A-type K+ channel RCK4, Raw3 channels operate in the positive voltage range.     

Nerve growth factor regulates the abundance and distribution of K+ channels in PC12 cells

We examined the effect of nerve growth factor (NGF) treatment on expression of a neuronal delayed rectifler K+ channel subtype, Kv2.1 (drk1), in PC12 cells. Anti-Kv2.1 antibodies recognized a single polypeptide population of M(r) = 132 kD in PC12 cell membranes, distinct from the more heterogeneous population found in adult rat brain. In response to NGF treatment, levels of Kv2.1 polypeptide in PC12 membranes increased fourfold. This increase in polypeptide levels could be seen within 12 h, and elevated levels were maintained for at least 6 d of continuous NGF treatment. RNase protection assays indicate that this increase in Kv2.1 protein occurs without an increase in steady state levels of Kv2.1 mRNA following NGF treatment. Immunofluorescent localization of the Kv2.1 polypeptide revealed plasma membrane-associated staining of cell bodies in both untreated and NGF- treated PC12 cells. In undifferentiated cells, intense staining is seen at sites of cell-cell and cell-substratum contact. In differentiated cells the most intense Kv2.1 staining is observed in neuritic growth cones. These studies show that in PC12 cells both the abundance and distribution of the Kv2.1 k+ channel are regulated by NGF, and suggest that PC12 cells provide a model for the selective expression of Kv2.1 in neuritic endings.     

Errata

Celecoxib is a drug designed to selectively inhibit COX-2, an inflammation-inducible cyclooxygenase isoform, over the constitutively expressed COX-1 isoform. In addition to this selective inhibition it is now known that celecoxib exerts a variety of effects on several types of ion channels, thus producing secondary physiological effects. In this work we demonstrate that at therapeutically relevant concentrations celecoxib interacts with Shab K+ channels specifically promoting a fast inactivation gating (without blocking the pore or significantly affecting other gating processes). At least two celecoxib molecules bind to each channel promoting a fast inactivation that develops from both open and closed states. Channel inactivation in turn causes a reduction of the size of IK. Taken together, our observations show that in addition to its intended therapeutic target celecoxib is a useful tool to further study the mechanism of Shab channel inactivation.     

Kv2.1/Kv9.3, a novel ATP-dependent delayed-rectifier K+ channel in oxygen-sensitive pulmonary artery myocytes.

The molecular structure of oxygen-sensitive delayed-rectifier K+ channels which are involved in hypoxic pulmonary artery (PA) vasoconstriction has yet to be elucidated. To address this problem, we identified the Shab K+ channel Kv2.1 and a novel Shab-like subunit Kv9.3, in rat PA myocytes. Kv9.3 encodes an electrically silent subunit which associates with Kv2.1 and modulates its biophysical properties. The Kv2.1/9.3 heteromultimer, unlike Kv2.1, opens in the voltage range of the resting membrane potential of PA myocytes. Moreover, we demonstrate that the activity of Kv2.1/Kv9.3 is tightly controlled by internal ATP and is reversibly inhibited by hypoxia. In conclusion, we propose that metabolic regulation of the Kv2.1/Kv9.3 heteromultimer may play an important role in hypoxic PA vasoconstriction and in the possible development of PA hypertension.     

Expression of ion channels and receptors in Xenopus oocytes using vaccinia virus.

The cytoplasmic injection of mRNA synthesized in vitro into Xenopus oocytes is widely used for heterologous expression of ion channels and neurotransmitter receptors. We report two new methods for expression of ion channels and receptors in oocytes using vaccinia virus (VV). 1) A recombinant VV carrying the Shaker H4 K+ channel cDNA driven by the VV P7.5 early promoter was injected into oocytes. 2) A recombinant VV containing the bacteriophage T7 RNA polymerase driven by the P7.5 promoter was coinjected along with plasmids containing a T7 promoter and cDNAs for channels and receptors. The functionally expressed proteins include a) voltage-gated ion channels: the Shaker H4 K+ channel and the rat brain IIA Na+ channel, b) a ligand-gated ion channel: the mouse muscle nicotinic acetylcholine receptor (AChR), and c) a G protein-coupled receptor: the rat brain 5HT1C receptor. After virus/cDNA injection into oocytes, these channels and receptors generally showed characteristics and expression levels similar to those observed in mRNA-injected oocytes. However, the AChR expressed at lower levels in virus/cDNA-injected oocytes than in mRNA-injected oocytes. Because our methods bypass mRNA synthesis, they are more rapid and convenient than the mRNA injection method. Potential applications to structure-function studies and expression cloning are discussed.     

Potassium channels from NG108-15 neuroblastoma-glioma hybrid cells. Primary structure and functional expression from cDNAs

The complete amino acid sequences of two potassium channel proteins from NG108-15 neuroblastoma-glioma hybrid cells have been deduced by cloning and sequencing the cDNAs. One of these proteins (NGK2) is structurally more closely related to the Drosophila Shaw gene product than to the Shaker and Shab gene products, whereas the other (NGK1) is identical with a rat brain potassium channel protein (BK2) which is more closely related to the Drosophila Shaker gene product. mRNAs derived from both the cloned cDNAs, when injected into Xenopus oocytes, direct the formation of functional potassium channels with properties of delayed rectifiers.     

THIK-1 and THIK-2, a novel subfamily of tandem pore domain K+ channels

Two cDNAs encoding novel K(+) channels, THIK-1 and THIK-2 (tandem pore domain halothane inhibited K(+) channel), were isolated from rat brain. The proteins of 405 and 430 amino acids were 58% identical to each other. Homology analysis showed that the novel channels form a separate subfamily among tandem pore domain K(+) channels. The genes of the human orthologs were identified as human genomic data base entries. They possess one intron each and were assigned to chromosomal region 14q24.1-14q24.3 (human (h) THIK-1) and 2p22-2p21 (hTHIK-2). In rat (r), THIK-1 (rTHIK-1) is expressed ubiquitously; rTHIK-2 expression was found in several tissues including brain and kidney. In situ hybridization of brain slices showed that rTHIK-2 is strongly expressed in most brain regions, whereas rTHIK-1 expression is more restricted. Heterologous expression of rTHIK-1 in Xenopus oocytes revealed a K(+) channel displaying weak inward rectification in symmetrical K(+) solution. The current was enhanced by arachidonic acid and inhibited by halothane. rTHIK-2 did not functionally express. Confocal microscopy of oocytes injected with green fluorescent protein-tagged rTHIK-1 or rTHIK-2 showed that both channel subunits are targeted to the outer membrane. However, coinjection of rTHIK-2 did not affect the currents induced by rTHIK-1, indicating that the two channel subunits do not form heteromers.     

Shaker, Shal, Shab, and Shaw express independent K+ current systems.

Although many K+ channel genes encoding homologous subunits have been cloned, a central question remains: how do these subunits associate to produce the diversity of K+ currents observed in living cells? Previous work has shown that different subunits encoded by the Shaker gene subfamily are able to form heteromultimers, which add to the diversity of currents. However, the unrestrained mixing of subunits from all genes to form hybrid channels would be undesirable for some cells that clearly require functionally discrete K+ currents. We show that Drosophila Shaker, Shal, Shab, and Shaw subunits form functional homomultimers, but that a molecular barrier to heteropolymerization is present. Coexpression of all four K+ channel systems does not alter their individual properties in any way. These experiments also demonstrate that multiple, independent A-current systems together with multiple, independent delayed rectifier systems can coexist in single cells.     

Cloning and functional expression of a liver isoform of the small conductance Ca2+-activated K+ channel SK3

Small conductance Ca2+-activated K+ (SK) channels have been cloned from mammalian brain, but little is known about the molecular characteristics of SK channels in nonexcitable tissues. Here, we report the isolation from rat liver of an isoform of SK3. The sequence of the rat liver isoform differs from rat brain SK3 in five amino acid residues in the NH3 terminus, where it more closely resembles human brain SK3. SK3 immunoreactivity was detectable in hepatocytes in rat liver and in HTC rat hepatoma cells. Human embryonic kidney (HEK-293) cells transfected with liver SK3 expressed 10 pS K+ channels that were Ca2+ dependent (EC(50) 630 nM) and were blocked by the SK channel inhibitor apamin (IC(50) 0.6 nM); whole cell SK3 currents inactivated at membrane potentials more positive than -40 mV. Notably, the Ca2+ dependence, apamin sensitivity, and voltage-dependent inactivation of SK3 are strikingly similar to the properties of hepatocellular and biliary epithelial SK channels evoked by metabolic stress. These observations raise the possibility that SK3 channels influence membrane K+ permeability in hepatobiliary cells during liver injury.     

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 calcium and ATP sensitive nonselective cation channel in the antiluminal membrane of rat cerebral capillary endothelial cells.

The patch-clamp technique was applied to the antiluminal membrane of freshly isolated capillaries of rat brain (blood-brain barrier). With 1.3 mM Ca2+ in the bath, excision of membrane patches evoked ion channels, which could not be observed in cell-attached mode. The channel was about equally permeable to Na+ and K+ ions, but not measurable permeable to Cl- and the divalent ions Ca2+ and Ba2+. The current-voltage curve was linear in the investigated voltage range (-80 mV to +80 mV), and the single-channel conductance was 31 +/- 2 pS (n = 22). The channel open probability was not dependent on the applied potential. Lowering of Ca2+ to 1 microM or below on the cytosolic side inactivated the channels, whereas addition of cytosolic ATP (1 mM) inhibited channel activity completely and reversibly. The channel was blocked by the inhibitor of nonselective cation channels in rat exocrine pancreas 3',5-dichlorodiphenylamine-2-carboxylic acid (DCDPC, 10 microM) and by the antiinflammatory drugs flufenamic acid (greater than 10 microM) and tenidap (100 microM), as well as by gadolinium (10 microM). Thus, these nonselective cation channels have many properties in common with similar channels observed in fluid secreting epithelia. The channel could be involved in the transport of K+ ions from brain to blood side.     

Light-dependent modulation of Shab channels via phosphoinositide depletion in Drosophila photoreceptors

The Drosophila phototransduction cascade transforms light into depolarizations that are further shaped by activation of voltage-dependent K+ (Kv) channels. In whole-cell recordings of isolated photoreceptors, we show that light selectively modulated the delayed rectifier (Shab) current. Shab currents were increased by light with similar kinetics to the light-induced current itself (latency approximately 20 ms), recovering to control values with a t(1/2) of approximately 60 s in darkness. Genetic disruption of PLCbeta4, responsible for light-induced PIP(2) hydrolysis, abolished this light-dependent modulation. In mutants of CDP-diaclyglycerol synthase (cds(1)), required for PIP(2) resynthesis, the modulation became irreversible, but exogenously applied PIP(2) restored reversibility. The modulation was accurately and reversibly mimicked by application of PIP(2) to heterologously expressed Shab channels in excised inside-out patches. The results indicate a functionally implemented mechanism of Kv channel modulation by PIP(2) in photoreceptors, which enables light-dependent regulation of signal processing by direct coupling to the phototransduction cascade.