Silent TWIK-1 potassium channels conduct monovalent cation currents

TWIK-1 two-pore domain K(+) channels generally produce nonmeasurable or very low levels of K(+) currents in heterologous expression systems under physiologically ionic conditions. Two controversial mechanisms have been proposed to account for this behavior: TWIK-1 K(+) channels are expressed in the cell surface but silenced by sumoylation at a lysine residue (TWIK-1 K274); constitutive and rapid internalization of TWIK-1 causes TWIK-1 channel silencing. Here we report that TWIK-1 K(+) channels heterologously expressed in Chinese hamster ovary cells, which are silent in physiological K(+) gradients, are able to conduct large monovalent cation currents when extracellular ionic conditions change. These results support the hypothesis that TWIK-1 K(+) channels are expressed in the cell surface but silent, and suggest that the TWIK-1 gating behavior rather than the lack of cell surface expression of TWIK-1 results in nondetectable TWIK-1 K(+) currents in heterologous expression systems.     

TWIK-2, a new weak inward rectifying member of the tandem pore domain potassium channel family

Potassium channels are found in all mammalian cell types, and they perform many distinct functions in both excitable and non-excitable cells. These functions are subserved by several different families of potassium channels distinguishable by primary sequence features as well as by physiological characteristics. Of these families, the tandem pore domain potassium channels are a new and distinct class, primarily distinguished by the presence of two pore-forming domains within a single polypeptide chain. We have cloned a new member of this family, TWIK-2, from a human brain cDNA library. Primary sequence analysis of TWIK-2 shows that it is most closely related to TWIK-1, especially in the pore-forming domains. Northern blot analysis reveals the expression of TWIK-2 in all human tissues assayed except skeletal muscle. Human TWIK-2 expressed heterologously in Xenopus oocytes is a non-inactivating weak inward rectifier with channel properties similar to TWIK-1. Pharmacologically, TWIK-2 channels are distinct from TWIK-1 channels in their response to quinidine, quinine, and barium. TWIK-2 is inhibited by intracellular, but not extracellular, acidification. This new clone reveals the existence of a subfamily in the tandem pore domain potassium channel family with weak inward rectification properties.     

Molecular cloning and functional expression of KCNQ5, a potassium channel subunit that may contribute to neuronal M-current diversity

We have isolated KCNQ5, a novel human member of the KCNQ potassium channel gene family that is differentially expressed in subregions of the brain and in skeletal muscle. When expressed in Xenopus oocytes, KCNQ5 generated voltage-dependent, slowly activating K(+)-selective currents that displayed a marked inward rectification at positive membrane voltages. KCNQ5 currents were insensitive to the K(+) channel blocker tetraethylammonium but were strongly inhibited by the selective M-current blocker linopirdine. Upon coexpression with the structurally related KCNQ3 channel subunit, current amplitudes increased 4-5-fold. Compared with homomeric KCNQ5 currents, KCNQ3/KCNQ5 currents also displayed slower activation kinetics and less inward rectification, indicating that KCNQ5 combined with KCNQ3 to form functional heteromeric channel proteins. This functional interaction between KCNQ5 and KCNQ3, a component of the M-channel, suggests that KCNQ5 may contribute to a diversity of heteromeric channels underlying native neuronal M-currents.     

Evidence for two-pore domain potassium channels in rat cerebral arteries

Little is known about the presence and function of two-pore domain K(+) (K(2P)) channels in vascular smooth muscle cells (VSMCs). Five members of the K(2P) channel family are known to be directly activated by arachidonic acid (AA). The purpose of this study was to determine 1) whether AA-sensitive K(2P) channels are expressed in cerebral VSMCs and 2) whether AA dilates the rat middle cerebral artery (MCA) by increasing K+ currents in VSMCs via an atypical K+ channel. RT-PCR revealed message for the following AA-sensitive K(2P) channels in rat MCA: tandem of P domains in weak inward rectifier K+ (TWIK-2), TWIK-related K+ (TREK-1 and TREK-2), TWIK-related AA-stimulated K+ (TRAAK), and TWIK-related halothane-inhibited K+ (THIK-1) channels. However, in isolated VSMCs, only message for TWIK-2 was found. Western blotting showed that TWIK-2 is present in MCA, and immunohistochemistry further demonstrated its presence in VSMCs. AA (10-100 microM) dilated MCAs through an endothelium-independent mechanism. AA-induced dilation was not affected by inhibition of cyclooxygenase, epoxygenase, or lipoxygenase or inhibition of classical K+ channels with 10 mM TEA, 3 mM 4-aminopyridine, 10 microM glibenclamide, or 100 microM Ba2+. AA-induced dilations were blocked by 50 mM K+, indicating involvement of a K+ channel. AA (10 microM) increased whole cell K+ currents in dispersed cerebral VSMCs. AA-induced currents were not affected by inhibitors of the AA metabolic pathways or blockade of classical K+ channels. We conclude that AA dilates the rat MCA and increases K+ currents in VSMCs via an atypical K+ channel that is likely a member of the K(2P) channel family.     

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.     

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.     

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.     

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.     

Identification and cloning of TWIK-originated similarity sequence (TOSS): a novel human 2-pore K channel principal subunit

We have identified and cloned a new member of the mammalian tandem pore domain K+ channel subunit family, TWIK-originated similarity sequence, from a human testis cDNA library. The 939 bp open reading frame encodes a 313 amino acid polypeptide with a calculated Mr of 33.7 kDa. Despite the same predicted topology, there is a relatively low sequence homology between TWIK-originated similarity sequence and other members of the mammalian tandem pore domain K+ channel subunit family group. TWIK-originated similarity sequence shares a low (< 30%) identity with the other mammalian tandem pore domain K+ channel subunit family group members and the highest identity (34%) with TWIK-1 at the amino acid level. Similar low levels of sequence homology exist between all members of the mammalian tandem pore domain K+ channel subunit family. Potential glycosylation and consensus PKC sites are present. Northern analysis revealed species and tissue-specific expression patterns. Expression of TWIK-originated similarity sequence is restricted to human pancreas, placenta and heart, while in the mouse, TWIK-originated similarity sequence is expressed in the liver. No functional currents were observed in Xenopus laevis oocytes or HEK293T cells, suggesting that TWIK-originated similarity sequence may be targeted to locations other than the plasma membrane or that TWIK-originated similarity sequence may represent a novel regulatory mammalian tandem pore domain K+ channel subunit family subunit.     

A Ba2+-resistant, acid-sensitive K+ conductance in Na+-absorbing H441 human airway epithelial cells

By analysis of whole cell membrane currents in Na(+)-absorbing H441 human airway epithelial cells, we have identified a K(+) conductance (G(K)) resistant to Ba(2+) but sensitive to bupivacaine or extracellular acidification. In polarized H441 monolayers, we have demonstrated that bupivacaine, lidocaine, and quinidine inhibit basolateral membrane K(+) current (I(Bl)) whereas Ba(2+) has only a weak inhibitory effect. I(Bl) was also inhibited by basolateral acidification, and, although subsequent addition of bupivacaine caused a further fall in I(Bl), acidification had no effect after bupivacaine, demonstrating that cells grown under these conditions express at least two different bupivacaine-sensitive K(+) channels, only one of which is acid sensitive. Basolateral acidification also inhibited short-circuit current (I(SC)), and basolateral bupivacaine, lidocaine, quinidine, and Ba(2+) inhibited I(SC) at concentrations similar to those needed to inhibit I(Bl), suggesting that the K(+) channels underlying I(Bl) are part of the absorptive mechanism. Analyses using RT-PCR showed that mRNA encoding several two-pore domain K(+) (K2P) channels was detected in cells grown under standard conditions (TWIK-1, TREK-1, TASK-2, TWIK-2, KCNK-7, TASK-3, TREK-2, THIK-1, and TALK-2). We therefore suggest that K2P channels underlie G(K) in unstimulated cells and so maintain the driving force for Na(+) absorption. Since this ion transport process is vital to lung function, K2P channels thus play an important but previously undocumented role in pulmonary physiology.     

Dexamethasone-induced up-regulation of two-pore domain K+ channel genes, TASK-1 and TWIK-2, in cultured human periodontal ligament fibroblasts

Two-pore domain K(+) channels are widely expressed in many types of cells, and have various important functions, especially maintaining the resting membrane potential. In the previous report, we have confirmed the presence of several kinds of two-pore domain K(+) channels in the periodontal ligament (PDL) fibroblasts. It is well known that dexamethasone (Dex) regulates the functions of various kinds of ion channels. In this work, we investigate if Dex affects the gene expressions of the two-pore domain K(+) channels in the PDL fibroblasts. We also examined the effects of other steroid hormones on the K(+) channels gene expression. The mRNA levels of two-pore domain K(+) channels in human PDL fibroblasts were examined in the presence or absence of Dex by RT-PCR. The effects of other steroid hormones (aldosterone, estrogen, 1α,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], and retinoic acid) were also examined. Dex significantly induced the expression of TASK-1 and TWIK-2 in mRNA levels in both a dose- and a time-dependent manner. The stimulatory effects of Dex were completely abolished by a glucocorticoid receptor antagonist. 1,25-(OH)(2)D(3) also increased the TASK-1 mRNA levels but had no effect on TWIK-2 expression. Dex, one of the potent glucocorticoid, probably have a protective role against external stimuli by maintaining the membrane potential of PDL fibroblasts through the up-regulation of TASK-1 and TWIK-2 K(+) channels.     

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.     

Enhancement of HERG K(+) currents by Cd(2+) destabilization of the inactivated state

We have studied the functional effects of extracellular Cd(2+) on human ether-a-go-go-related gene (HERG) encoded K(+) channels. Low concentrations (10-200 &mgr;M) of extracellular Cd(2+) increased outward currents through HERG channels; 200 &mgr;M Cd(2+) more than doubled HERG currents and altered current kinetics. Cd(2+) concentrations up to 200 &mgr;M did not change the voltage dependence of channel activation, but shifted the voltage dependence of inactivation to more depolarized membrane potentials. Cd(2+) concentrations >/=500 &mgr;M shifted the voltage dependence of channel activation to more positive potentials. These results are consistent with a somewhat specific ability of Cd(2+) to destabilize the inactivated state. We tested the hypothesis that channel inactivation is essential for Cd(2+)-induced increases in HERG K(+) currents, using a double point mutation (G628C/S631C) that diminishes HERG inactivation (Smith, P. L., T. Baukrowitz, and G. Yellen. 1996. Nature (Lond.). 379:833-836). This inactivation-removed mutant is insensitive to low concentrations of Cd(2+). Thus, Cd(2+) had two distinct effects on HERG K(+) channels. Low concentrations of Cd(2+) caused relatively selective effects on inactivation, resulting in a reduction of the apparent rectification of the channel and thereby increasing HERG K(+) currents. Higher Cd(2+) concentrations affected activation gating as well, possibly by a surface charge screening mechanism or by association with a lower affinity site.     

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.     

Identification and functional characterization of zebrafish K(2P)10.1 (TREK2) two-pore-domain K(+) channels

Two-pore-domain potassium (K(2P)) channels mediate K(+) background currents that stabilize the resting membrane potential and contribute to repolarization of action potentials in excitable cells. The functional significance of K(2P) currents in cardiac electrophysiology remains poorly understood. Danio rerio (zebrafish) may be utilized to elucidate the role of cardiac K(2P) channels in vivo. The aim of this work was to identify and functionally characterize a zebrafish otholog of the human K(2P)10.1 channel. K(2P)10.1 orthologs in the D. rerio genome were identified by database analysis, and the full zK(2P)10.1 coding sequence was amplified from zebrafish cDNA. Human and zebrafish K(2P)10.1 proteins share 61% identity. High degrees of conservation were observed in protein domains relevant for structural integrity and regulation. K(2P)10.1 channels were heterologously expressed in Xenopus oocytes, and currents were recorded using two-electrode voltage clamp electrophysiology. Human and zebrafish channels mediated K(+) selective background currents leading to membrane hyperpolarization. Arachidonic acid, an activator of hK(2P)10.1, induced robust activation of zK(2P)10.1. Activity of both channels was reduced by protein kinase C. Similar to its human counterpart, zK(2P)10.1 was inhibited by the antiarrhythmic drug amiodarone. In summary, zebrafish harbor K(2P)10.1 two-pore-domain K(+) channels that exhibit structural and functional properties largely similar to human K(2P)10.1. We conclude that the zebrafish represents a valid model to study K(2P)10.1 function in vivo.     

Identification of a novel tetramerization domain in large conductance K(ca) channels

More than 50 genes are known to encode K(+) channel monomers and can coassemble to form hetero-tetrameric K(+) channels. However, only a subset of possible monomer combinations come together to form functional ion channels. The assembly and tetramerization of appropriate channel monomers is mediated by association domains (ADs). To identify such domains in human large-conductance Ca(2+)-activated K(+) channels (hSlo1), we screened hSlo1 domains for self-association using yeast two-hybrid assays. Putative ADs were subjected to functional assays in Xenopus oocytes and further characterized by coprecipitation, native gel electrophoresis, and sucrose density gradient centrifugation assays. This led to the identification of a single intracellular association domain localized near the channel pore and required for channel function. We conclude that this novel tetramerization domain, referred to as BK-T1, promotes the assembly of hSlo1 monomers into functional K(Ca) channels.     

Protein kinase A is central for forward transport of two-pore domain potassium channels K2P3.1 and K2P9.1

Acid-sensitive two-pore domain potassium channels (K2P3.1 and K2P9.1) play key roles in both physiological and pathophysiological mechanisms, the most fundamental of which is control of resting membrane potential of cells in which they are expressed. These background "leak" channels are constitutively active once expressed at the plasma membrane, and hence tight control of their targeting and surface expression is fundamental to the regulation of K(+) flux and cell excitability. The chaperone protein, 14-3-3, binds to a critical phosphorylated serine in the channel C termini of K2P3.1 and K2P9.1 (Ser(393) and Ser(373), respectively) and overcomes retention in the endoplasmic reticulum by βCOP. We sought to identify the kinase responsible for phosphorylation of the terminal serine in human and rat variants of K2P3.1 and K2P9.1. Adopting a bioinformatic approach, three candidate protein kinases were identified: cAMP-dependent protein kinase, ribosomal S6 kinase, and protein kinase C. In vitro phosphorylation assays were utilized to determine the ability of the candidate kinases to phosphorylate the channel C termini. Electrophysiological measurements of human K2P3.1 transiently expressed in HEK293 cells and cell surface assays of GFP-tagged K2P3.1 and K2P9.1 enabled the determination of the functional implications of phosphorylation by specific kinases. All of our findings support the conclusion that cAMP-dependent protein kinase is responsible for the phosphorylation of the terminal serine in both K2P3.1 and K2P9.1.     

Overexpression of monomeric and multimeric GIRK4 subunits in rat atrial myocytes removes fast desensitization and reduces inward rectification of muscarinic K(+) current (I(K(ACh))). Evidence for functional homomeric GIRK4 channels

K(+) channels composed of G-protein-coupled inwardly rectifying K(+) channel (GIRK) (Kir3.0) subunits are expressed in cardiac, neuronal, and various endocrine tissues. They are involved in inhibiting excitability and contribute to regulating important physiological functions such as cardiac frequency and secretion of hormones. The functional cardiac (K((ACh))) channel activated by G(i)/G(o)-coupled receptors such as muscarinic M(2) or purinergic A(1) receptors is supposed to be composed of the subunits GIRK1 and GIRK4 in a heterotetrameric (2:2) fashion. In the present study, we have manipulated the subunit composition of the K((ACh)) channels in cultured atrial myocytes from hearts of adult rats by transient transfection of vectors encoding for GIRK1 or GIRK4 subunits or GIRK4 concatemeric constructs and investigated the effects on properties of macroscopic I(K(ACh)). Transfection with a GIRK1 vector did not cause any measurable effect on properties of I(K(ACh)), whereas transfection with a GIRK4 vector resulted in a complete loss in desensitization, a reduction of inward rectification, and a slowing of activation. Transfection of myocytes with a construct encoding for a concatemeric GIRK4(2) subunit had similar effects on desensitization and inward rectification. Following transfection of a tetrameric construct (GIRK4(4)), these changes in properties of I(K(ACh)) were still observed but were less pronounced. Heterologous expression in Chinese hamster ovary cells and human embryonic kidney 293 cells of monomeric, dimeric, and tetrameric GIRK4 resulted in robust currents activated by co-expressed A(1) and M(2) receptors, respectively. These data provide strong evidence that homomeric GIRK4 complexes form functional G(beta)gamma gated ion channels and that kinetic properties of GIRK channels, such as activation rate, desensitization, and inward rectification, depend on subunit composition.     

New structure and function in plant K+ channels: KCO1, an outward rectifier with a steep Ca2+ dependency.

Potassium (K+) channels mediating important physiological functions are characterized by a common pore-forming (P) domain. We report the cloning and functional analysis of the first higher plant outward rectifying K+ channel (KCO1) from Arabidopsis thaliana. KCO1 belongs to a new class of ''two-pore'' K+ channels recently described in human and yeast. KCO1 has four putative transmembrane segments and tandem calcium-binding EF-hand motifs. Heterologous expression of KCO1 in baculovirus-infected insect (Spodoptera frugiperda) cells resulted in outwardly rectifying, K+-selective currents elicited by depolarizing voltage pulses in whole-cell measurements. Activation of KCO1 was strongly dependent on the presence of nanomolar concentrations of cytosolic free Ca2+ [Ca2+]cyt. No K+ currents were detected when [Ca2+]cyt was adjusted to <150 nM. However, KCO1 strongly activated at increasing [Ca2+]cyt, with a saturating activity observed at approximately 300 nM [Ca2+]cyt. KCO1 single channel analysis on excised membrane patches, resulting in a single channel conductance of 64 pS, confirmed outward rectification as well as Ca2+-dependent activation. These data suggest a direct link between calcium-mediated signaling processes and K+ ion transport in higher plants. The identification of KCO1 as the first plant K+ outward channel opens a new field of structure-function studies in plant ion channels.     

Contribution of P2X1 receptor intracellular basic residues to channel properties

The intracellular amino and carboxy termini of P2X receptors have been shown to contribute to the regulation of ATP evoked currents. In this study we produced, and expressed in Xenopus oocytes, individual alanine point mutants of positively charged amino acids (eight lysine, seven arginine and one histidine) in the intracellular domains of the human P2X1 receptor. The majority of these mutations had no effect on the amplitude, time-course or rectification of ATP evoked currents. In contrast the mutant K367A was expressed at normal levels at the cell surface however ATP evoked currents were reduced by >99% and desensitised more rapidly demonstrating a role of K367 in channel regulation. This is similar to that previously described for T18A mutant channels. Co-expression of T18A and K367A mutant P2X1 receptors produced larger ATP evoked responses than either mutant alone and suggests that these amino and carboxy terminal regions interact to regulate channel function.