PubMed Health⌕ Search

Biomedical subjects

D E Logothetis

Publications and source records attributed to D E Logothetis.

At least 19 recordsLinked to original sources
PubMed2001

Cloning and characterization of G protein-gated inward rectifier K+ channel (GIRK1) isoforms from heart and brain.

A G protein-gated inward rectifier potassium (K+) channel (GIRK1a) has been cloned from different tissues (Kubo et al., 1993b; Dascal et al., 1993). Here we report the cloning of three additional novel isoforms of GIRK1a from rat atria and and one from human brain. These isoform cDNAs code for proteins that have identical N-termini, M1-H5-M2 (predicted transmembrane and pore domains), and post-M2 amino acid regions to GIRK1a (1-501 amino acids), but they have shorter C-termini (GIRK1b (1-309), GIRK1c (1-308), GIRK1d (1-235), and GIRK1e (1-253). These results indicated that isoforms were generated by alternative splicing and partial genomic analysis confirmed the presence of exons and introns in the rat GIRK1 gene. RNase protection analysis and immunoblot analysis indicated that the isoforms were expressed in both rat atria and brain but at lower levels versus GIRK1a. The physiological role that the isoforms may play in atrial and brain physiology remains to be determined.

Alternative Splicing↗
PubMed2000

Visualization of a functional Galpha q-green fluorescent protein fusion in living cells. Association with the plasma membrane is disrupted by mutational activation and by elimination of palmitoylation sites, but not be activation mediated by receptors or AlF4-.

To investigate how G protein alpha subunit localization is regulated under basal and activated conditions, we inserted green fluorescent protein (GFP) into an internal loop of Galpha(q). alpha(q)-GFP stimulates phospholipase C in response to activated receptors and inhibits betagamma-dependent activation of basal G protein-gated inwardly rectifying K(+) currents as effectively as alpha(q) does. Association of alpha(q)-GFP with the plasma membrane is reduced by mutational activation and eliminated by mutation of the alpha(q) palmitoylation sites, suggesting that alpha(q) must be in the inactive, palmitoylated state to be targeted to this location. We tested the effects of activation by receptors and by AlF(4)(-) on the localization of alpha(q)-GFP in cells expressing both alpha(q)-GFP and a protein kinase Cgamma-red fluorescent protein fusion that translocates to the plasma membrane in response to activation of G(q). In cells that clearly exhibit protein kinase Cgamma-red fluorescent protein translocation responses, relocalization of alpha(q)-GFP is not observed. Thus, under conditions associated with palmitate turnover and betagamma dissociation, alpha(q)-GFP remains associated with the plasma membrane. These results suggest that upon reaching the plasma membrane alpha(q) receives an anchoring signal in addition to palmitoylation and association with betagamma, or that during activation, one or both of these factors continues to retain alpha(q) in this location.

Aluminum Compounds↗
PubMed2000

Glycosylation of GIRK1 at Asn119 and ROMK1 at Asn117 has different consequences in potassium channel function.

GIRK (G protein-gated inward rectifier K(+) channel) proteins play critical functional roles in heart and brain physiology. Using antibodies directed to either GIRK1 or GIRK4, site-directed mutagenesis, and specific glycosidases, we have investigated the effects of glycosylation in the biosynthesis and heteromerization of these proteins expressed in oocytes. Both GIRK1 and GIRK4 have one extracellular consensus N-glycosylation site. Using chimeras between GIRK1 and GIRK4 as well as a GIRK1 N-glycosylation mutant, we report that GIRK1 was glycosylated at Asn(119), whereas GIRK4 was not glycosylated at Asn(132). GIRK1 membrane-spanning domain 1 was required for optimal glycosylation at Asn(119) because a chimera that contained GIRK4 membrane-spanning domain 1 significantly reduced the addition of a carbohydrate structure at this site. This finding may partly account for the reason that GIRK4 is not glycosylated at Asn(132), either as a homomer or when coexpressed with GIRK1. When the GIRK1(N119Q) mutant was coexpressed with GIRK4, the biophysical properties of the heteromeric channel and the magnitude of the agonist-induced currents were similar to those of controls. Thus, N-glycosylation of GIRK1 at Asn(119) does not appear to affect its physical association with GIRK4, the routing of the heteromer to the cell surface, or heteromeric channel function, unlike the dramatic functional effects of N-glycosylation of ROMK1 at Asn(117) (Schwalbe, R. A., Wang, Z., Wible, B. A., and Brown, A. M. (1995) J. Biol. Chem. 270, 15336-15340).

Amidohydrolases↗
PubMed2000

Receptor-mediated hydrolysis of plasma membrane messenger PIP2 leads to K+-current desensitization.

Phosphatidylinositol bisphosphate (PIP2) directly regulates functions as diverse as the organization of the cytoskeleton, vesicular transport and ion channel activity. It is not known, however, whether dynamic changes in PIP2 levels have a regulatory role of physiological importance in such functions. Here, we show in both native cardiac cells and heterologous expression systems that receptor-regulated PIP2 hydrolysis results in desensitization of a GTP-binding protein-stimulated potassium current. Two receptor-regulated pathways in the plasma membrane cross-talk at the level of these channels to modulate potassium currents. One pathway signals through the betagamma subunits of G proteins, which bind directly to the channel. Gbetagamma subunits stabilize interactions with PIP2 and lead to persistent channel activation. The second pathway activates phospholipase C (PLC) which hydrolyses PIP2 and limits Gbetagamma-stimulated activity. Our results provide evidence that PIP2 itself is a receptor-regulated second messenger, downregulation of which accounts for a new form of desensitization.

Acetylcholine↗
PubMed1999

Distinct specificities of inwardly rectifying K(+) channels for phosphoinositides.

Activation of several inwardly rectifying K(+) channels (Kir) requires the presence of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). The constitutively active Kir2.1 (IRK1) channels interact with PtdIns(4,5)P(2) strongly, whereas the G-protein activated Kir3.1/3.4 channels (GIRK1/GIRK4), show only weak interactions with PtdIns(4,5)P(2). We investigated whether these inwardly rectifying K(+) channels displayed distinct specificities for different phosphoinositides. IRK1, but not GIRK1/GIRK4 channels, showed a marked specificity toward phosphates in the 4,5 head group positions. GIRK1/GIRK4 channels were activated with a similar efficacy by PtdIns(3,4)P(2), PtdIns(3,5)P(2), PtdIns(4,5)P(2), and PtdIns(3,4,5)P(3). In contrast, IRK1 channels were not activated by PtdIns(3,4)P(2) and only marginally by high concentrations of PtdIns(3,5)P(2). Similarly, high concentrations of PtdIns(3,4,5)P(3) were required to activate IRK1 channels. For either channel, PtdIns(4)P was much less effective than PtdIns(4,5)P(2), whereas PtdIns was inactive. In contrast to the dependence on the position of phosphates of the phospholipid head group, GIRK1/GIRK4, but not IRK1 channel activation, showed a remarkable dependence on the phospholipid acyl chains. GIRK1/GIRK4 channels were activated most effectively by the natural arachidonyl stearyl PtdIns(4,5)P(2) and much less by the synthetic dipalmitoyl analog, whereas IRK1 channels were activated equally by dipalmitoyl and arachidonyl stearyl PtdIns(4,5)P(2). Incorporation of PtdInsP(2) into the membrane is necessary for activation, as the short chain water soluble diC(4) PtdIns(4,5)P(2) did not activate either channel, whereas activation by diC(8) PtdIns(4, 5)P(2) required high concentrations.

Animals↗
PubMed1999

Gating of G protein-sensitive inwardly rectifying K+ channels through phosphatidylinositol 4,5-bisphosphate.

Several inwardly rectifying K+ channels show an ATP-dependent rundown of their activity. Hydrolysis of ATP is required for maintenance of channel activity. G protein-gated inwardly rectifying K+ (GIRK) channels also depend on ATP hydrolysis for gating by sodium ions or the [beta][gamma] subunits of G proteins (Sui et al. 1998). Strong evidence suggests that phosphatidylinositol 4, 5-bisphosphate (PIP2), synthesized via the hydrolysis of ATP, is absolutely required for channel gating (Sui et al. 1998; Huang et al. 1998). Interestingly, Huang and colleagues (Huang et al. 1998) showed that G[beta][gamma] subunits (the [beta][gamma] subunits of GTP-binding proteins) caused a stabilization of channel-PIP2 interactions, suggesting that G[beta][gamma] subunits may gate the channel through PIP2. Ho & Murrell-Lagnado (1999b) recently identified an aspartate residue responsible for gating these K+ channels. Ho & Murrell-Lagnado (1999a) in this issue of The Journal of Physiology present evidence that sodium ions also stabilize channel-PIP2 interactions. They suggest that Na+ effectively neutralizes a negatively charged residue, somehow promoting interactions of the channel with PIP2. These results on the mechanism of Na+ action are in close agreement with recently published work from our group. Zhang et al. (1999) showed that two C-terminal cytoplasmic arginine residues, which interact with PIP2, are localized next to the identified aspartate residue that is responsible for the Na+ effects on gating. Thus, the implication from the results of these three studies (Huang et al. 1998; Zhang et al. 1999; Ho & Murrell-Lagnado, 1999a) is that stabilization of channel-PIP2 interactions may be a common mechanism for gating GIRK channels by molecules as different as Na+ or the G[beta][gamma] subunits (see Fig. 1). These results raise many interesting questions on how modulation of channel-PIP2 interactions may lead to channel gating. Are the channel-PIP2 sites that are stabilized by G[beta][gamma] subunits shared with those that are affected by Na+? Na+ seems to act by screening the electrostatic effects that the aspartate residue exerts on the nearby PIP2-interacting arginines. How does G[beta][gamma] binding lead to stabilization of channel- PIP2 interactions? Is the Na+ sensitivity of the channel used physiologically and if so how does it relate to signalling through G proteins? Structural data for these channels and in particular for their cytoplasmic portions, which are critical for interactions with PIP2, will greatly aid our molecular understanding of the conformations needed for channel gating. Finally, although the dependence of GIRK channel activity on PIP2 is clear, it is not known yet whether the levels of PIP2 needed for channel gating are constant or under regulatory control. Answers to these and many other such questions are likely to shed light on the mechanism by which PIP2 itself serves as an important regulator of the activity of membrane proteins.

Ion Channel Gating↗
PubMed1999

Identification of a potassium channel site that interacts with G protein betagamma subunits to mediate agonist-induced signaling.

Activation of heterotrimeric GTP-binding (G) proteins by their coupled receptors, causes dissociation of the G protein alpha and betagamma subunits. Gbetagamma subunits interact directly with G protein-gated inwardly rectifying K+ (GIRK) channels to stimulate their activity. In addition, free Gbetagamma subunits, resulting from agonist-independent dissociation of G protein subunits, can account for a major component of the basal channel activity. Using a series of chimeric constructs between GIRK4 and a Gbetagamma-insensitive K+ channel, IRK1, we have identified a critical site of interaction of GIRK with Gbetagamma. Mutation of Leu339 to Glu within this site impaired agonist-induced sensitivity and decreased binding to Gbetagamma, without removing the Gbetagamma contribution to basal currents. Mutation of the corresponding residue in GIRK1 (Leu333) resulted in a similar phenotype. Both the GIRK1 and GIRK4 subunits contributed equally to the agonist-induced sensitivity of the heteromultimeric channel. Thus, we have identified a channel site that interacts specifically with Gbetagamma subunits released through receptor stimulation.

Binding Sites↗
PubMed1999

Activation of inwardly rectifying K+ channels by distinct PtdIns(4,5)P2 interactions.

Direct interactions of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) with inwardly rectifying potassium channels are stronger with channels rendered constitutively active by binding to PtdIns(4,5)P2, such as IRK1, than with G-protein-gated channels (GIRKs). As a result, PtdIns(4,5)P2 alone can activate IRK1 but not GIRKs, which require extra gating molecules such as the beta gamma subunits of G proteins or sodium ions. Here we identify two conserved residues near the inner-membrane interface of these channels that are critical in interactions with PtdIns(4,5)P2. Between these two arginines, a conservative change of isoleucine residue 229 in GIRK4 to the corresponding leucine found in IRK1 strengthens GIRK4-PtdIns(4,5)P2 interactions, eliminating the need for extra gating molecules. A negatively charged GIRK4 residue, two positions away from the most strongly interacting arginine, mediates stimulation of channel activity by sodium by strengthening channel-PtdIns(4,5)P2 interactions. Our results provide a mechanistic framework for understanding how distinct gating mechanisms of inwardly rectifying potassium channels allow these channels to subserve their physiological roles.

Amino Acid Sequence↗
PubMed1999

Synergistic activation of G protein-gated inwardly rectifying potassium channels by the betagamma subunits of G proteins and Na(+) and Mg(2+) ions.

Native and recombinant G protein-gated inwardly rectifying potassium (GIRK) channels are directly activated by the betagamma subunits of GTP-binding (G) proteins. The presence of phosphatidylinositol-bis-phosphate (PIP(2)) is required for G protein activation. Formation (via hydrolysis of ATP) of endogenous PIP(2) or application of exogenous PIP(2) increases the mean open time of GIRK channels and sensitizes them to gating by internal Na(+) ions. In the present study, we show that the activity of ATP- or PIP(2)-modified channels could also be stimulated by intracellular Mg(2+) ions. In addition, Mg(2+) ions reduced the single-channel conductance of GIRK channels, independently of their gating ability. Both Na(+) and Mg(2+) ions exert their gating effects independently of each other or of the activation by the G(betagamma) subunits. At high levels of PIP(2), synergistic interactions among Na(+), Mg(2+), and G(betagamma) subunits resulted in severalfold stimulated levels of channel activity. Changes in ionic concentrations and/or G protein subunits in the local environment of these K(+) channels could provide a rapid amplification mechanism for generation of graded activity, thereby adjusting the level of excitability of the cells.

Adenosine Triphosphate↗
PubMed1998

Characterization of a Ca2+-activated K+ current in insulin-secreting murine betaTC-3 cells.

1. The whole-cell perforated-patch recording mode was used to record a Ca2+-dependent K+ current (IK(Ca)) in mouse betaTC-3 insulin-secreting cells. 2. Depolarizing voltage steps (to potentials where Ca2+ currents are activated) evoked a slowly activating, outward current, which exhibited a slow deactivation (in seconds) upon subsequent hyperpolarization. 3. This current was shown to increase with progressively longer depolarizing voltage steps. It could be reversibly abolished by the removal of Ca2+ from the external medium or by application of Ca2+ channel blockers, such as Cd2+ and nifedipine. It was concluded that the depolarization-evoked current was activated by Ca2+. 4. Variations in external K+ concentration led to shifts in the reversal potential of the Ca2+-dependent current as predicted by the Nernst equation for a K+-selective current. 5. The Ca2+-activated K+ current was insensitive to external TEA (10 mM), a concentration sufficient to block the large-conductance Ca2+-dependent (maxi-KCa) channel in beta-cells. It was also insensitive to apamin, tubocurarine and scyllatoxin (leiurotoxin I), specific blockers of small-conductance KCa channels. 6. The current was blocked by quinine, a non-specific KCa channel blocker and, surprisingly, by charybdotoxin (ChTX; 100 nM) but not iberiotoxin, a charybdotoxin analogue, which blocks the maxi-KCa channel. It was sensitive to block by clotrimazole and could be potently and reversibly potentiated by micromolar concentrations of niflumic acid. Thus, the current exhibited unique pharmacological characteristics, not conforming to the known KCa channel classes. 7. The ChTX-sensitive KCa channel was permeable to Tl+, K+, Rb+ and NH4+ but not Cs+ ions. 8. The ChTX-sensitive IK(Ca) could be activated by the muscarinic agonists in the presence or absence of external Ca2+, presumably by releasing Ca2+ from internal stores. 9. Acutely isolated porcine islet cells also exhibited a slow IK(Ca) resembling that described in betaTC-3 cells in kinetic properties, insensitivity to TEA (5 mM) and sensitivity to quinidine, an analogue of quinine. The porcine IK(Ca), however, was not sensitive to block by 100-200 nM ChTX. It is likely, that species differences account for pharmacological differences between the mouse and porcine slow IK(Ca).

Animals↗
PubMed1998

Activation of the atrial KACh channel by the betagamma subunits of G proteins or intracellular Na+ ions depends on the presence of phosphatidylinositol phosphates.

The betagamma subunits of GTP-binding proteins (Gbetagamma) activate the muscarinic K+ channel (KACh) in heart by direct binding to both of its component subunits. KACh channels can also be gated by internal Na+ ions. Both activation mechanisms show dependence on hydrolysis of intracellular ATP. We report that phosphatidylinositol 4,5-bisphosphate (PIP2) mimics the ATP effects and that depletion or block of PIP2 retards the stimulatory effects of Gbetagamma subunits or Na+ ions on channel activity, effects that can be reversed by restoring PIP2. Thus, regulation of KACh channel activity may be crucially dependent on PIP2 and phosphatidylinositol signaling. These striking functional results are in agreement with in vitro biochemical studies on the PIP2 requirement for Gbetagamma stimulation of G protein receptor kinase activity, thus implicating phosphatidylinositol phospholipids as a potential control point for Gbetagamma-mediated signal transduction.

Adenosine Triphosphate↗
PubMed1997

Probing the G-protein regulation of GIRK1 and GIRK4, the two subunits of the KACh channel, using functional homomeric mutants.

In heart, G-protein-activated channels are complexes of two homologous proteins, GIRK1 and GIRK4. Expression of either protein alone results in barely active or non-active channels, making it difficult to assess the individual contribution of each subunit to the channel complex. The residue Phe137, located within the H5 region of GIRK1, is critical to the synergy between GIRK1 and GIRK4 (Chan, K. W., Sui, J. L., Vivaudou, M., and Logothetis, D. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14193-14198). By modifying this residue or the matching residue of GIRK4, Ser143, we have been able to generate mutant proteins that produced large inwardly rectifying, G-protein-modulated currents when expressed alone in Xenopus oocytes. The enhanced activity of the heterologous expression of each of two active mutants, GIRK1(F137S) and GIRK4(S143T), was not caused by association with an endogenous oocyte channel subunit, and these mutants did not display apparent differences in the ability to localize to the cell surface compared with their wild-type counterparts. When these functional mutant channels were compared individually with wild-type heteromeric channels, they responded with only small differences to a number of maneuvers involving coexpression with muscarinic receptors, G-protein betagamma subunits, wild-type or mutated G-protein alpha subunits, and active protomers of pertussis toxin. These experiments, which confirmed the crucial, though not exclusive, role of Gbetagamma in regulating channel activity, demonstrated that GIRK1(F137S) and GIRK4(S143T), and by extrapolation their wild-type counterparts, interact in a qualitatively similar way with G-protein subunits. These findings suggest that functionally important sites of interaction with G-proteins are likely to be located within the homologous regions of GIRK1 and GIRK4 rather than within the divergent terminal regions. They also raise the question of the functional advantage of a heteromeric over homomeric design for G-protein-gated channels.

Animals↗
PubMed1997

Specific regions of heteromeric subunits involved in enhancement of G protein-gated K+ channel activity.

Heterologous coexpression of recombinant, G protein-gated, inwardly rectifying K+ (GIRK) channel subunits has yielded large currents, severalfold greater than those obtained from expression of the individual subunits. Such current enhancement has been obtained from coexpression of the inactive GIRK1 subunit with the low activity GIRK2-5 subunits in Xenopus oocytes. Using deletion and chimeric constructs, we now report the identification of a C-terminal region unique to GIRK1 and a larger central region of GIRK4 highly homologous to GIRK1, both of which are critical for production of large currents. Chimeras containing these two regions produced homomeric channels, exhibiting currents severalfold greater than those from either wild-type subunit alone. G protein regulation of such chimeric channel currents resembled that of wild-type currents. Green fluorescent protein-tagged channels showed that the amount of chimeric channel expressed on the oocyte cell surface was similar to its wild-type counterpart, suggesting that the enhanced activity was not due to differences in relative levels of expression but rather to the coexistence of the chimeric regions. Single-channel recordings of the active chimeras exhibited patterns of activities with open-time kinetics and conductance characteristics representative of those of GIRK4, indicating that the presence of the GIRK1 C-terminal region caused an increase in the frequency of channel openings without affecting their duration.

Amino Acid Sequence↗
PubMed1997

A calcium-dependent chloride current in insulin-secreting beta TC-3 cells.

Ca(2+)-dependent conductances have been hypothesized to play a role in the bursting pattern of electrical activity of insulin-secreting beta cells in response to high plasma glucose. A Maxi K+ channel has received the most attention, while a low-conductance Ca(2+)-activated K+ current has also been identified. We used an increasingly popular beta cell model system, the beta TC-3 cell line, and the perforated-patch technique to describe the properties of a novel Ca(2+)-dependent Cl- current [ICl(Ca)] in insulin-secreting pancreatic beta cells. The reported ICl(Ca) could be activated under physiological Ca2+ concentrations and is the first of its kind to be described in pancreatic insulin-secreting cells. We found that long depolarizing steps above -20 mV elicited an outward current which showed slow inward relaxation upon repolarization to negative membrane potentials. Both the outward currents and the inward tails showed dependence on Ca2+ influx: their current/voltage (I/V) relations followed that of the "L-like" Ca2+ current (ICa) present in these cells; they were blocked completely by the removal of external Ca2+ or application of Cd2+ at concentrations sufficient for complete block of ICa; and their magnitude increased with the depolarizing step duration. Moreover, the inward tail decayed monoexponentially with a time constant which at voltages negative to activation of ICa showed a weak linear voltage dependence, while at voltages positive to activation of ICa it followed the voltage dependence of ICa. This Ca(2+)-dependent current reversed at -21.5 mV and when the external Cl- concentration was reduced from 159 mM to 62 mM the reversal potential shifted by approximately +20 mV as predicted by the Nernst relation for a Cl(-)-selective current. Cl- channel blockers such as DIDS (100 microM) and niflumic acid (100 microM) blocked this current. We concluded that this current was a Ca(2+)-dependent Cl- current [ICl(Ca)]. From substitution of the external Cl- with various monovalent anions and from the reversal potentials we obtained the following permeability sequence for ICl(Ca): I- > NO3- > Br- > Cl- > Acetate.

Animals↗
PubMed1996

Control of channel activity through a unique amino acid residue of a G protein-gated inwardly rectifying K+ channel subunit.

G protein-gated inwardly rectifying K+ (GIRK) channels, which are important regulators of membrane excitability both in heart and brain, appear to function as heteromultimers. GIRK1 is unique in the GIRK channel family in that although it is by itself inactive, it can associate with the other family members (GIRK2-GIRK5) to enhance their activity and alter their single-channel characteristics. By generating a series of chimeras, we identified a phenylalanine residue, F137, in the pore region of GIRK1 that critically controls channel activity. F137 is found only in GIRK1, while the remaining GIRK channels possess a conserved serine residue in the analogous position. The single-point mutant GIRK4(S143F) behaved as a GIRK1 analog, forming multimers with GIRK2, GIRK4, or GIRK5 channels that exhibited prolonged single-channel open-time duration and enhanced activity compared with that of homomultimers. Expression of the corresponding GIRK1 (F137S) mutant alone resulted in appreciable channel activity with novel characteristics that was further enhanced upon coexpression with other GIRK subunits. Thus, although the F137 residue renders the GIRK1 subunit inactive, when combined with other GIRK heteromeric partners it alters their gating and contributes to their enhanced activity.

Amino Acid Sequence↗
PubMed1996

A recombinant inwardly rectifying potassium channel coupled to GTP-binding proteins.

GTP-binding (G) proteins have been shown to mediate activation of inwardly rectifying potassium (K+) channels in cardiac, neuronal and neuroendocrine cells. Here, we report functional expression of a recombinant inwardly rectifying channel which we call KGP (or hpKir3.4), to signify that it is K+ selective, G-protein-gated and isolated from human pancreas. KGP expression in Xenopus oocytes resulted in sizeable basal (or agonist-independent) currents while coexpression with a G-protein-linked receptor, yielded additional agonist-induced currents. Coexpression of KGP and hGIRK1 (a human brain homolog of GIRK1/Kir3.1) produced much larger basal currents than those observed with KGP or hGIRK1 alone, and upon coexpression with receptor, similarly large agonist-induced currents could be obtained. Pertussis toxin treatment significantly diminished agonist-dependent currents due to either KGP or KGP/hGIRK1 expression. Interestingly, PTX also significantly reduced basal KGP or KGP/hGIRK1 currents, suggesting that basal activity is largely the result of G-protein gating as well. When the two channels were coexpressed with receptor, the relative increase in current elicited by agonist was similar whether KGP and hGIRK1 were expressed alone or together. When in vitro translated or when expressed in Xenopus oocytes or CHO mammalian cells, KGP gave rise to a nonglycosylated 45-kD protein. Antibodies directed against either KGP or hGIRK1 coprecipitated both proteins coexpressed in oocytes, providing evidence for the heteromeric assembly of the two channels and suggesting that the current potentiation seen with coexpression of the two channel subunits is due to specific interactions between them. An endogenous oocyte protein similar in size to KGP was also coprecipitated with hGIRK1.

Acetylcholine↗
PubMed1996

Na+ activation of the muscarinic K+ channel by a G-protein-independent mechanism.

Muscarinic potassium channels (KACh) are composed of two subunits, GIRK1 and GIRK4 (or CIR), and are directly gated by G proteins. We have identified a novel gating mechanism of KACh, independent of G-protein activation. This mechanism involved functional modification of KACh which required hydrolysis of physiological levels of intracellular ATP and was manifested by an increase in the channel mean open time. The ATP-modified channels could in turn be gated by intracellular Na+, starting at approximately 3 mM with an EC50 of approximately 40 mM. The Na(+)-gating of KACh was operative both in native atrial cells and in a heterologous system expressing recombinant channel subunits. Block of the Na+/K+ pump (e.g., by cardiac glycosides) caused significant activation of KACh in atrial cells, with a time course similar to that of Na+ accumulation and in a manner indistinguishable from that of Na(+)-mediated activation of the channel, suggesting that cardiac glycosides activated KACh by increasing intracellular Na+ levels. These results demonstrate for the first time a direct effect of cardiac glycosides on atrial myocytes involving ion channels which are critical in the regulation of cardiac rhythm.

Adenosine Triphosphate↗