Overexpression of KCNJ3 gene splice variants affects vital parameters of the malignant breast cancer cell line MCF-7 in an opposing manner.

BACKGROUND: Overexpression the KCNJ3, a gene that encodes subunit 1 of G-protein activated inwardly rectifying K(+) channel (GIRK1) in the primary tumor has been found to be associated with reduced survival times and increased lymph node metastasis in breast cancer patients. METHODS: In order to survey possible tumorigenic properties of GIRK1 overexpression, a range of malignant mammary epithelial cells, based on the MCF-7 cell line that permanently overexpress different splice variants of the KCNJ3 gene (GIRK1a, GIRK1c, GIRK1d and as a control, eYFP) were produced. Subsequently, selected cardinal neoplasia associated cellular parameters were assessed and compared. RESULTS: Adhesion to fibronectin coated surface as well as cell proliferation remained unaffected. Other vital parameters intimately linked to malignancy, i.e. wound healing, chemoinvasion, cellular velocities / motilities and angiogenesis were massively affected by GIRK1 overexpression. Overexpression of different GIRK1 splice variants exerted differential actions. While GIRK1a and GIRK1c overexpression reinforced the affected parameters towards malignancy, overexpression of GIRK1d resulted in the opposite. Single channel recording using the patch clamp technique revealed functional GIRK channels in the plasma membrane of MCF-7 cells albeit at very low frequency. DISCUSSION: We conclude that GIRK1d acts as a dominant negative constituent of functional GIRK complexes present in the plasma membrane of MCF-7 cells, while overexpression of GIRK1a and GIRK1c augmented their activity. The core component responsible for the cancerogenic action of GIRK1 is apparently presented by a segment comprising aminoacids 235-402, that is present exclusively in GIRK1a and GIRK1c, but not GIRK1d (positions according to GIRK1a primary structure). CONCLUSIONS: The current study provides insight into the cellular and molecular consequences of KCNJ3 overexpression in breast cancer cells and the mechanism upon clinical outcome in patients suffering from breast cancer.

Subunit stoichiometry of heterologously expressed G-protein activated inwardly rectifying potassium channels analysed by fluorescence intensity ratio measurement.

The Xenopus laevis oocyte expression system offers the unique opportunity to heterologously express many proteins simultaneously and to control the expression level for every protein individually. By using the expression of fusion constructs of variants of the green fluorescence protein (eCFP, eGFP and eYFP) with GIRK1 and GIRK4 subunits and measuring the respective fluorescence intensity ratios (FIRs) of the expressed proteins by confocal laser scan microscopy, we were able to measure the amount of each of the individual subunits expressed. At equal amounts of injected RNAs encoding GIRK1 and GIRK4, we found that approximately 2.2 GIRK4 subunits per 1 GIRK1 subunit appeared at the surface of the oocyte, suggesting the coexistence of homooligomeric GIRK4 complexes with heterooligomeric GIRK1/GIRK4 complexes. Interestingly, when the ratio of injected RNA is increased in favour of GIRK1, the subunit stoichiometry changes accordingly until, at a RNA ratio of 25:1 (GIRK1/GIRK4), the subunit stoichiometry is shifted towards a protein complex with 3:1 stoichiometry (GIRK1/GIRK4). In parallel, the amount of GIRK1 protein appearing at the surface gets greatly reduced, supporting previous studies that showed that the GIRK1 subunit needs assembly with GIRK4 for surface localization. By using a genetically encoded marker for the endoplasmic reticulum (ER), we were able to show that the subunit stoichiometry in regions of the ER, which are located directly below the plasma membrane, closely resembles that observed directly at the surface. Generally, our study reveals that the subunit stoichiometry of GIRK1/GIRK4 channels in the Xenopus laevis oocyte expression system depends to a great extent on the molar ratio of the different RNAs injected.

G protein-gated inwardly rectifying potassium channels are targets for volatile anesthetics.

G protein-gated inwardly rectifying potassium channels (GIRKs) are a family of homo- and hetero-oligomeric K(+) channels composed of different subunits (GIRK1 to 4 in mammals). GIRK4 and GIRK1 are found mainly in the atrium, whereas neuronal cells predominantly express the GIRK1, GIRK2, and GIRK3 isoforms. When activated, GIRK channels slow the firing rate of atrial myocytes and neuronal cells. Because of their key role in controlling excitability, we investigated the influence of a prototypic anesthetic, halothane, on GIRK channels of different subunit composition expressed in Xenopus laevis oocytes. Halothane enhanced background currents through hetero-oligomeric GIRK1/GIRK4 and homo-oligomeric GIRK1(F137S) channels but not through homo-oligomeric GIRK2 channels. This activation of basal current did not depend on the presence of coexpressed G protein-coupled receptors but instead required the presence of G(beta/gamma). In contrast to basal GIRK currents, the agonist-induced GIRK current (via coexpressed m2 muscarinic receptors) was inhibited by halothane. For GIRK1/GIRK4 and GIRK1(F137S) channels this inhibition was most pronounced at low concentrations of the anesthetic (0.1-0.3 mM) and occurred also when channels had been activated by guanosine-5'-O-(3-thio)triphosphate. This inhibition, however, was overridden by high concentrations of halothane (0.9 mM) and augmentation of the agonist-induced current was observed. This increase in agonist-induced current was never seen with GIRK2 homo-oligomeric channels. Agonist-induced currents mediated by GIRK2 channels were always inhibited by halothane with an IC(50) value of approximately 60 microM. These data suggest a direct interaction of halothane with GIRK channels.

S-nitrosation controls gating and conductance of the alpha 1 subunit of class C L-type Ca(2+) channels.

Modulation of smooth muscle, L-type Ca(2+) channels (class C, Ca(V)1.2b) by thionitrite S-nitrosoglutathione (GSNO) was investigated in the human embryonic kidney 293 expression system at the level of whole-cell and single-channel currents. Extracellular administration of GSNO (2 mM) rapidly reduced whole-cell Ba(2+) currents through channels derived either by expression of alpha1C-b or by coexpression of alpha1C-b plus beta2a and alpha2-delta. The non-thiol nitric oxide (NO) donors 2,2-diethyl-1-nitroso-oxhydrazin (2 mM) and 3-morpholinosydnonimine-hydrochloride (2 mM), which elevated cellular cGMP levels to a similar extent as GSNO, failed to affect Ba(2+) currents significantly. Intracellular administration of copper ions, which promote decomposition of the thionitrite, antagonized its inhibitory effect, and loading of cells with high concentrations of dithiothreitol (2 mM) prevented the effect of GSNO on alpha1C-b channels. Intracellular loading of cells with oxidized glutathione (2 mM) affected neither alpha1C-b channel function nor their modulation by GSNO. Analysis of single-channel behavior revealed that GSNO inhibited Ca(2+) channels mainly by reducing open probability. The development of GSNO-induced inhibition was associated with the transient occurrence of a reduced conductance state of the channel. Our results demonstrate that GSNO modulates the alpha1 subunit of smooth muscle L-type Ca(2+) channels by an intracellular mechanism that is independent of NO release and stimulation of guanylyl cyclase. We suggest S-nitrosation of intracellularly located sulfhydryl groups as an important determinant of Ca(2+) channel gating and conductance.

Heterologous facilitation of G protein-activated K(+) channels by beta-adrenergic stimulation via cAMP-dependent protein kinase.

To investigate possible effects of adrenergic stimulation on G protein-activated inwardly rectifying K(+) channels (GIRK), acetylcholine (ACh)-evoked K(+) current, I(KACh), was recorded from adult rat atrial cardiomyocytes using the whole cell patch clamp method and a fast perfusion system. The rise time of I(KACh ) was 0. 4 +/- 0.1 s. When isoproterenol (Iso) was applied simultaneously with ACh, an additional slow component (11.4 +/- 3.0 s) appeared, and the amplitude of the elicited I(KACh) was increased by 22.9 +/- 5.4%. Both the slow component of activation and the current increase caused by Iso were abolished by preincubation in 50 microM H89 (N-[2-((p -bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, a potent inhibitor of PKA). This heterologous facilitation of GIRK current by beta-adrenergic stimulation was further studied in Xenopus laevis oocytes coexpressing beta(2)-adrenergic receptors, m(2 )-receptors, and GIRK1/GIRK4 subunits. Both Iso and ACh elicited GIRK currents in these oocytes. Furthermore, Iso facilitated ACh currents in a way, similar to atrial cells. Cytosolic injection of 30-60 pmol cAMP, but not of Rp-cAMPS (a cAMP analogue that is inhibitory to PKA) mimicked the beta(2)-adrenergic effect. The possibility that the potentiation of GIRK currents was a result of the phosphorylation of the beta-adrenergic receptor (beta(2)AR) by PKA was excluded by using a mutant beta(2)AR in which the residues for PKA-mediated modulation were mutated. Overexpression of the alpha subunit of G proteins (Galpha(s)) led to an increase in basal as well as agonist-induced GIRK1/GIRK4 currents (inhibited by H89). At higher levels of expressed Galpha(s), GIRK currents were inhibited, presumably due to sequestration of the beta/gamma subunit dimer of G protein. GIRK1/GIRK5, GIRK1/GIRK2, and homomeric GIRK2 channels were also regulated by cAMP injections. Mutant GIRK1/GIRK4 channels in which the 40 COOH-terminal amino acids (which contain a strong PKA phosphorylation consensus site) were deleted were also modulated by cAMP injections. Hence, the structural determinant responsible is not located within this region. We conclude that, both in atrial myocytes and in Xenopus oocytes, beta-adrenergic stimulation potentiates the ACh-evoked GIRK channels via a pathway that involves PKA-catalyzed phosphorylation downstream from beta(2)AR.

IK.ACh activation by arachidonic acid occurs via a G-protein-independent pathway mediated by the GIRK1 subunit.

The molecular target of arachidonic-acid-derived metabolites, serving as second messengers that activate atrial acetylcholine-activated potassium current (IK.ACh) in addition to G-protein beta/gamma subunits (Gbeta/gamma), is unknown. Co-expression of two isoforms of G-protein-activated, inwardly rectifying potassium channels (GIRKs) in oocytes of Xenopus laevis revealed that these heterologous co-expressed GIRKs, which are responsible for the formation of IK.ACh in the atrium, are activated by arachidonic acid metabolites, like their counterparts in atrial cells. The expression of homooligomeric GIRK1(F137S) and GIRK4wt channels revealed that this activatory mechanism is specific to the GIRKI subunit. Sequestrating available Gbeta/gamma by overexpression of C-betaARK (a Gbeta/gamma binding protein) failed to abolish the activation of GIRK currents by arachidonic acid. From our experiments we conclude that the GIRKI subunit itself is the molecular target for regulation of GIRK channels by arachidonic acid metabolites.

Isoform diversity and modulation of sodium channels by protein kinases.

Although voltage-dependent sodium channels from the brain have been realized as targets for regulatory protein phosphorylation since the first isolation of sodium channels as proteins, the functional role of phosphoprotein formation has been obscured until recently. The review summarizes progress on the modulation of voltage-dependent sodium channels by serine/threonine protein kinases, i.e. PKA and PKC. Divergent modulation is discussed in context with divergent primary structure of the alpha-subunit.

The cardiac acetylcholine-activated, inwardly rectifying K+-channel subunit GIRK1 gives rise to an inward current induced by free oxygen radicals.

Reactive oxygen species (ROS) play a crucial role in pathophysiology of the cardiovascular system. The present study was designed to analyze the redox sensitivity of G-protein-activated inward rectifier K+ (GIRK) channels, which control cardiac contractility and excitability. GIRK1 subunits were heterologously expressed in Xenopus laevis oocytes and the resulting K+ currents were measured with the two-electrode voltage clamp technique. Oxygen free radicals generated by the hypoxanthine/xanthine oxidase system led to a marked increase in the current through GIRK channels, termed superoxide-induced current (I(SO)). Furthermore, I(SO) did not depend on G-protein-dependent activation of GIRK currents by coexpressed muscarinic m2-receptors, but could also be observed when no agonist was present in the bathing solution. Niflumic acid at a concentration of 0.5 mmol/l did not abolish I(SO), whereas 100 micromol/l Ba2+ attenuated I(SO) completely. Catalase (10(6) i.u./l) failed to suppress I(SO), whereas H2O2 concentration was kept close to zero, as measured by chemiluminescence. Hence, we conclude that O2*- or a closely related species is responsible for I(SO) induction. Our results demonstrate a significant redox sensitivity of GIRK1 channels and suggest redox-activation of G-protein-activated inward rectifier K+ channels as a key mechanism in oxidative stress-associated cardiac dysfunction.

Trp proteins form store-operated cation channels in human vascular endothelial cells.

Members of the Trp protein family have been suggested as the structural basis of store-operated cation conductances. With this study, we provide evidence for the expression of three isoforms of Trp (hTrp1, 3 and 4) in human umbilical vein endothelial cells (HUVEC). The role of Trp proteins in store regulation of endothelial membrane conductances was tested by expression of an N-terminal fragment of hTrp3 (N-TRP) which exerts a dominant negative effect on Trp channel function presumably due to suppression of channel assembly. Depletion of intracellular Ca2+ stores with IP3 (100 microM) or thapsigargin (100 nM) induced a substantial cation conductance in sham-transfected HUVEC as well as in HUVEC transfected with hTrp3. In contrast, HUVEC transfected with N-TRP failed to exhibit store-operated currents. Our results suggest the involvement of Trp related proteins in the store-operated cation conductance of human vascular endothelial cells.

Ion permeation through a G-protein activated (GIRK1/GIRK5) inwardly rectifying potassium channel.

In order to further investigate a G-protein activated inwardly rectifying potassium channel subunit, GIRK1 was expressed in Xenopus oocytes (where it coassembles with the endogenous GIRK5). The mechanism underlying ion permeation and rectification were measured in isolated inside-out patches. Single channel current amplitudes under symmetrical K+ concentrations at different holding potentials were evaluated. Inward-rectification of K+-currents through open GIRK1/GIRK5 channels was removed by washing out polyamines and Mg2+ ions. We developed a simple 'two-sites-three-barrier' (2S3B) Eyring rate theory model of K+ ion permeation for GIRK1/GIRK5 channels. The resulting optimized parameter-set will be used as a working model for subsequent investigation regarding K+ permeation process through the GIRK1/GIRK5 channel.

A C-terminal peptide of the GIRK1 subunit directly blocks the G protein-activated K+ channel (GIRK) expressed in Xenopus oocytes.

1. In order to find out the functional roles of cytosolic regions of a G protein-activated, inwardly rectifying potassium channel subunit we studied block of GIRK channels, expressed in Xenopus laevis oocytes, by synthetic peptides in isolated inside-out membrane patches. 2. A peptide (DS6) derived from the very end of the C-terminus of GIRK1 reversibly blocked GIRK activity with IC50 values of 7.9 +/- 2.0 or 3.5 +/- 0.5 micrograms ml-1 (corresponding to 3.7 +/- 0.9 or 1.7 +/- 0.2 mumol l-1) for GIRK1/GIRK5 or GIRK1/GIRK4 channels, respectively. 3. Dose dependency studies of GIRK activation by purified beta gamma subunits of the G protein (G beta gamma) showed that DS6 block of GIRK channels is not the result of competition of the peptide with functional GIRK channels for the available G beta gamma. 4. Burst duration of GIRK channels was reduced, whereas long closed times between bursts were markedly increased, accounting for the channel block observed. 5. Block by the DS6 peptide was slightly voltage dependent, being stronger at more negative potentials. 6. These data support the hypothesis that the distal part of the carboxy-terminus of GIRK1 is a part of the intrinsic gate that keeps GIRK channels closed in the absence of G beta gamma.

Modulation of the human cardiac sodium channel alpha-subunit by cAMP-dependent protein kinase and the responsible sequence domain.

1. In order to investigate the modulation of human hH1 sodium channel alpha-subunits by cAMP-dependent protein kinase (PKA), the channel was expressed in oocytes of Xenopus laevis. 2. Cytosolic injection of cAMP, as well as of SP-cyclic 3',5'-hydrogen phosphorothioate adenosine triethylammonium salt (SP-cAMPS, the S-diastereoisomeric configuration of the compound with respect to the phosphorus atom), resulted in a marked and significant increase in peak sodium current (INa,p). Cytosolic injections of RP-cyclic 3',5'-hydrogen phosphorothioate adenosine triethylammonium salt (RP-cAMPS; a compound inhibitory to PKA) had no effect on peak current. 3. Kinetic parameters of steady-state activation, inactivation and recovery from inactivation were unchanged following stimulation of PKA activity, but a 42 +/- 5% (mean +/- S.E.M.) increase in maximal sodium conductance (delta gmax) could account for the observed increase in INa,p. 4. A set of chimerical sodium channels made from portions of the human cardiac hH1 alpha-subunit and the rat skeletal muscle SkM1 alpha-subunit (which is not affected by PKA stimulation) was generated. These were used to localize the structural determinant in the hH1 sequence responsible for PKA modulation of hH1. From our data we conclude that the effects of PKA on hH1 are conferred by the large cytosolic loop interconnecting transmembrane domains I and II, which is not conserved among sodium channel subtypes.

Inhibition of an inwardly rectifying K+ channel by G-protein alpha-subunits.

Cholinergic muscarinic, serotonergic, opioid and several other G-protein-coupled neurotransmitter receptors activate inwardly rectifying K+ channels of the GIRK family, slowing the heartbeat and decreasing the excitability of neuronal cells. Inhibitory modulation of GIRKs by G-protein-coupled receptors may have important implications in cardiac and brain physiology. Previously G alpha and G beta gamma subunits of heterotrimeric G proteins have both been implicated in channel opening, but recent studies attribute this role primarily to the G beta gamma dimer that activates GIRKs in a membrane-delimited fashion, probably by direct binding to the channel protein. We report here that free GTP gamma S-activated G alpha i 1, but not G alpha i 2 or G alpha i 3, potently inhibits G beta 1 gamma 2-induced GIRK activity in excised membrane patches of Xenopus oocytes expressing GIRK1. High-affinity but partial inhibition is produced by G alpha s-GTP gamma S. G alpha i 1-GTP gamma S also inhibits G beta 1 gamma 2-activated GIRK in atrial myocytes. Antagonistic interactions between G alpha and G beta gamma may be among the mechanisms determining specificity of G protein coupling to GIRKs.

Serotonin and protein kinase C modulation of a rat brain inwardly rectifying K+ channel expressed in xenopus oocytes.

In Xenopus laevis oocytes injected with rat brain poly(A)+ RNA, perfusion with a high-K+ solution (96 mM KCl) generated an inward current (IHK) which was absent in water-injected oocytes. Part of IHK was blocked by low concentrations of Ba2+ (half-maximal inhibitory concentration, IC50: 4.2 +/- 0.5 microM). When serotonin (5-HT) was applied to these oocytes a transient inward oscillating Cl- current arising from activation of Ca2+ -dependent Cl- channels, ICl (Ca), was observed. When this response decayed, a 30% reduction of IHK could be detected. Electrophysiological characterization of the K+ channel down-modulated by 5-HT revealed that it is an inward rectifier. Anti-sense suppression experiments revealed that the 5-HT2C receptor mediates the down-modulatory effect of 5-HT. The nature of the modulatory pathway was investigated by application of phorbol esters and intracellular injection of protein kinase C (PKC) inhibitors, ethylenebis (oxonitrilo)tetraacetate (EGTA) and inositol 1,4,5-trisphosphate. The results demonstrate that PKC is responsible for the down-modulatory effect.

Modulation of cardiac sodium channel isoform by cyclic AMP dependent protein kinase does not depend on phosphorylation of serine 1504 in the cytosolic loop interconnecting transmembrane domains III and IV.

Both the neuronal IIA as well as the cardiac SkM2 isoform of the pore forming alpha-subunit of voltage dependent sodium channels are modulated by Protein Kinase A. While alphaIIA becomes attenuated upon PKA stimulation, alphaSkM2 becomes upregulated. PKC dependent phosphorylation of a serine, located in the highly conserved cytoplasmatic region between the third and the fourth transmembraneous domain has been found to be a prerequisite for PKA modulation of the alphaIIA isoform. We used site-directed mutagenesis, expression in Xenopus laevis oocytes and the two-electrode voltage clamp technique to test, whether phosphorylation of the corresponding serine in alphaSkM2 is required for the PKA modulation of also the cardiac isoform. The results clearly indicate that serine 1504 does not play a significant role in the PKA modulation of the cardiac sodium channel isoform, further underlining the differential modulation of the two isoforms by identical signal transduction cascades.

Distribution and localization of a G protein-coupled inwardly rectifying K+ channel in the rat.

The cellular distribution of the mRNA of the inwardly rectifying K+ channel KGA (GIRK1) was investigated in rat tissue by in situ hybridization. KGA was originally cloned from the heart and represents the first G protein-activated K+ channel identified. It is expressed in peripheral tissue solely in the atrium, but not in the ventricle, skeletal muscle, lung and kidney. In the central nervous system KGA is most prominently expressed in the Ammon's horn and dentate gyrus of the hippocampus, neocortical layers II-VI, cerebellar granular layer, olfactory bulb, anterior pituitary, thalamic nuclei and several distinct nuclei of the lower brainstem. The abundant expression of KGA in many CNS neurons supports its important role as a major target channel for G protein mediated receptor function.

Voltage clamping of Xenopus laevis oocytes utilizing agarose-cushion electrodes.

Two-electrode voltage clamping of expressed ion channels in intact oocytes of the South African clawed frog Xenopus laevis has been refined to allow stable, low-resistance electrical access to the cytosol (50-800 k omega). Glass microelectrodes were filled with a cushion of 1% agarose at their tips to prevent KCl leakage (agarose-cushion electrodes). Insertion of these electrodes into X. laevis oocytes yielded stable preparations for periods of more than 1 h with a stable input resistance of 1-4 M omega. Furthermore, a simple modification of the voltage-clamp circuit (charging compensator) is described that increases the flexibility of arrangements for differential recording of the membrane potential in order to subtract voltage drops across a series resistance. The result is a considerable increase in the practically attainable speed of the voltage clamp with the conventional two-electrode arrangement. The performance of the charging compensator was tested on an equivalent circuit that simulates the oocyte and electrodes. In addition, the combination of agarose-cushion electrodes and the charging compensator was tested on oocytes expressing Shaker H4 currents. The fidelity of the voltage-clamp circuit was also verified by measuring the membrane potential with additional independent microelectrodes connected to a differential amplifier, independent of the two-electrode voltage clamp system. The system described here will be useful for ion channel studies in X. laevis oocytes requiring long-term recordings and/or measurements of large, fast ion currents.

Mechanism of modulation of single sodium channels from skeletal muscle by the beta 1-subunit from rat brain.

We studied the molecular mechanism of the rat skeletal muscle alpha-subunit (alpha microI) gating kinetics modulation by the brain beta 1-subunit by heterologous expression of single sodium channels from alpha microI and beta 1 in Xenopus laevis oocytes. Coexpression of beta 1 reduced mean open time at -10 mV to approximately 21% when compared to channels expressed by alpha microI alone. Channels formed by alpha microI exerted multiple openings per depolarization, which occurred in bursts, in contrast to the channels formed by the alpha microI/beta 1 complex that opened in average only once per depolarizing voltage pulse. Macroscopic current decay (mcd), as evidenced by reconstructed open probability vs. time (po(t)), was greatly accelerated by beta 1, closely resembling mcd of sodium currents from native skeletal muscle. Generally po(t) was larger for channels expressed from the pure alpha microI subunit. From our single channel data we conclude that beta 1 accelerates the inactivation process of the sodium channel complex.

Beta-adrenergic modulation of currents produced by rat cardiac Na+ channels expressed in Xenopus laevis oocytes.

In Xenopus oocytes coexpressing beta 2-adrenergic receptors and the rat cardiac alpha SkM2 Na+ channel, superfusion with 10 microM isoproterenol led to modest (approximately 30%) increases in peak Na+ inward current. Intracellular injection of cAMP and of protein kinase A (PKA) catalytic subunit reproduced this increase, showing that the second messenger pathway involves PKA dependent phosphorylation. Coexpression of the Na+ channel beta 1 subunit had no influence on the modulation. The modulation had little or no effect upon Na+ current waveforms, steady-state activation, steady-state activation, steady-state inactivation, or recovery from both fast and slow inactivation; but maximum Na+ conductance was increased. Mutation of the five major consensus PKA phosphorylation sites on alpha SkM2 did not abolish the observed effect. In parallel experiments, beta-adrenergic stimulation of the neuronal alpha IIA Na+ channel subunit led to an attenuation of Na+ current. It is concluded that (i) the alpha SkM2 subunit might be directly phosphorylated by PKA, but at serine/threonine residue(s) in a cryptic phosphorylation site(s); or that (ii) the modulation might also be mediated by phosphorylation of another, as yet unknown protein(s). The divergent modulation of neuronal and cardiac Na+ channel alpha-subunits suggests that differential physiological modulation by identical second messenger pathways is the evolutionary basis for the isoform diversity within this protein family.

Atrial G protein-activated K+ channel: expression cloning and molecular properties.

Activity of several ion channels is controlled by heterotrimeric GTP-binding proteins (G proteins) via a membrane-delimited pathway that does not involve cytoplasmic intermediates. The best studied example is the K+ channel activated by muscarinic agonists in the atrium, which plays a crucial role in regulating the heartbeat. To enable studies of the molecular mechanisms of activation, this channel, denoted KGA, was cloned from a rat atrium cDNA library by functional coupling to coexpressed serotonin type 1A receptors in Xenopus oocytes. KGA displays regions of sequence homology to other inwardly rectifying channels as well as unique regions that may govern G-protein interaction. The expressed KGA channel is activated by serotonin 1A, muscarinic m2, and delta-opioid receptors via G proteins. KGA is activated by guanosine 5'-[gamma-thio]triphosphate in excised patches, confirming activation by a membrane-delimited pathway, and displays a conductance equal to that of the endogenous channel in atrial cells. The hypothesis that similar channels play a role in neuronal inhibition is supported by the cloning of a nearly identical channel (KGB1) from a rat brain cDNA library.