Overexpression of beta 1 and beta 2 adrenergic receptors in rat atrial myocytes. Differential coupling to G protein-gated inward rectifier K(+) channels via G(s) and G(i)/o.

G protein-activated inwardly rectifying K(+) (GIRK) channels, expressed in atrial myocytes, various neurons, and endocrine cells, represent the paradigmatic target of beta gamma subunits released from activated heterotrimeric G proteins. These channels contribute to physiological slowing of cardiac frequency and synaptic inhibition. They are activated by beta gamma dimers released upon stimulation of receptors coupled to pertussis toxin-sensitive G proteins (G(i/o)), whereas beta gamma released from G(s) do not converge on the channel subunits. This is in conflict with the finding that dimeric combinations of various beta and gamma subunits can activate GIRK channels with little specificity. In the present study, we have overexpressed the major subtypes of cardiac beta-adrenergic receptors (beta(1)-AR and beta(2)-AR) in atrial myocytes by transient transfection. Whereas in native cells beta-adrenergic stimulation with isoproterenol failed to induce measurable GIRK current, robust currents were recorded from myocytes overexpressing either beta(1)-AR or beta(2)-AR. Whereas the beta(2)-AR-induced current showed the same sensitivity to pertussis toxin as the current evoked by the endogenous G(i/o)-coupled muscarinic M(2) receptor, isoproterenol-activated currents were insensitive to pertussis toxin treatment in beta(1)-AR-overexpressing myocytes. In contrast to a recent publication (Leaney, J. L., Milligan, G., and Tinker, A. (2000) J. Biol. Chem. 275, 921-929), sizable GIRK currents could also be activated by isoproterenol when the signaling pathway was reconstituted by transient transfection in two different standard cell lines (Chinese hamster ovary and HEK293). These results demonstrate that specificity of receptor-G protein signaling can be disrupted by overexpression of receptors. Moreover, the alpha subunit of heterotrimeric G proteins does not confer specificity to G beta gamma-mediated signaling.

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.

Depletion of phosphatidylinositol 4,5-bisphosphate by activation of phospholipase C-coupled receptors causes slow inhibition but not desensitization of G protein-gated inward rectifier K+ current in atrial myocytes.

G protein-gated inwardly rectifier K+ current in atrial myocytes (I(K(ACh))) upon stimulation with acetylcholine (ACh) shows a fast desensitizing component (t(1/2) approximately 5 s). After washout of ACh, I(K(ACh)) recovers from fast desensitization within < 30 s. A recent hypothesis suggests that fast desensitization is caused by depletion of phosphatidylinositol 4,5-bisphosphate (PtIns(4,5)P(2)), resulting from costimulation of phospholipase C (PLC)-coupled M3 receptors (M3AChR). The effects of stimulating two established PLC-coupled receptors, alpha-adrenergic and endothelin (ET(A)), on I(K(ACh)) were studied in rat atrial myocytes. Stimulation of these receptors caused activation of I(K(ACh)) and inhibition of the M2AChR-activated current. In myocytes loaded with GTPgammaS (guanosine 5'-3-O-(thio)triphosphate), causing stable activation of I(K(ACh)), inhibition via alpha-agonists and ET-1 was studied in isolation. Stimulation of either type of receptor under this condition, via G(q/11), caused a slow inhibition (t(1/2) approximately 50 s) by about 70%. No comparable effect on GTPgammaS-activated I(K(ACh)) was induced by ACh, suggesting that PLC-coupled M3AChRs are not functionally expressed in rat myocytes, which was supported by the finding that M3AChR transcripts were not detected by reverse transcriptase-polymerase chain reaction in identified atrial myocytes. Supplementing the pipette solution with PtIns(4,5)P(2) significantly reduced inhibition of I(K(ACh)) but had no effect on fast desensitization. From these data it is concluded that stimulation of PLC-coupled receptors causes slow inhibition of I(K(ACh)) by depletion of PtIns(4,5)P(2), whereas fast desensitization of I(K(ACh)) is not related to PtIns(4,5)P(2) depletion. As muscarinic stimulation by ACh does not exert inhibition of I(K(ACh)) comparable to stimulation of alpha(1)- and ET(A) receptors, expression of functional PLC-coupled muscarinic receptors in rat atrial myocytes is unlikely.

Overexpressed A(1) adenosine receptors reduce activation of acetylcholine-sensitive K(+) current by native muscarinic M(2) receptors in rat atrial myocytes.

In adult rat atrial myocytes, muscarinic acetylcholine (ACh)-sensitive K(+) current activated by a saturating concentration of adenosine (I(K(ACh),(Ado))) via A(1) receptors (A(1)Rs) amounts to only 30% of the current activated by a saturating concentration of ACh (I(K(ACh),(ACh))) via muscarinic M(2) receptors. The half-time of activation of I(K(ACh),(Ado)) on a rapid exposure to agonist was approximately 4-fold longer than that of I(K(ACh),(ACh)). Furthermore, I(K(ACh),(Ado)) never showed fast desensitization. To study the importance of receptor density for A(1)R-I(K(ACh),(Ado)) signaling, adult atrial myocytes in vitro were transfected with cDNA encoding for rat brain A(1)R and enhanced green fluorescent protein (EGFP) as a reporter. Whole-cell current was measured on days 3 and 4 after transfection. Time-matched cells transfected with only the EGFP vector served as controls. In approximately 30% of EGFP-positive cells (group I), the density of I(K(ACh),(Ado)) was increased by 72%, and its half-time of activation was reduced. Density and kinetic properties of I(K(ACh),(ACh)) were not affected in this fraction. In approximately 70% of transfection-positive myocytes (group II), the density of I(K(ACh),(ACh)) was significantly reduced, its activation was slowed, and the fast desensitizing component was lost. Adenosine-induced currents were larger in group II than in group I, their activation rate was further increased, and a fast desensitizing component developed. These data indicate that in native myocytes the amplitude and activation kinetics of I(K(ACh),(Ado)) are limited by the expression of A(1)R. Overexpression of A(1)R negatively interferes with signal transduction via the muscarinic M(2) receptor-linked pathway, which might reflect a competition of receptors with a common pool of G proteins. Negative interference of an overexpressed receptor with physiological regulation of a target protein by a different receptor should be considered in attempts to use receptor overexpression for gene therapy.

Antisense oligonucleotides against receptor kinase GRK2 disrupt target selectivity of beta-adrenergic receptors in atrial myocytes.

K+ channels composed of GIRK subunits are predominantly expressed in the heart and various regions of the brain. They are activated by betagamma-subunits released from pertussis toxin-sensitive G-proteins coupled to different seven-helix receptors. In rat atrial myocytes, activation of K(ACh) channels is strictly limited to receptors coupled to pertussis toxin-sensitive G-proteins. Upon treatment of myocytes with antisense oligodesoxynucleotides against GRK2, a receptor kinase with Gbetagamma binding sites, in a fraction of cells, K(ACh) channels can be activated by beta-adrenergic receptors. Sensitivity to beta-agonist is insensitive to pertussis toxin treatment. These findings demonstrate a potential role of Gbetagamma binding proteins for target selectivity of G-protein-coupled receptors.

Activation of muscarinic K+ current by beta-adrenergic receptors in cultured atrial myocytes transfected with beta1 subunit of heterotrimeric G proteins.

Muscarinic K+ channels (IK(ACh)) in native atrial myocytes are activated by betagamma subunits of pertussis toxin (Ptx)-sensitive heterotrimeric G proteins coupled to different receptors. betagamma subunits of Ptx-insensitive Gs, coupled to beta-adrenergic receptors, do not activate native IK(ACh). In atrial myocytes from adult rats transfected with rat brain beta1 subunit IK(ACh) can be activated by stimulation of beta-adrenergic receptors using isoprenaline. This effect is insensitive to Ptx. These findings demonstrate for the first time promiscuous (Ptx-insensitive) coupling of Gsbetagamma to GIRK channels in their native environment.

Non-receptor-mediated activation of IK(ATP) and inhibition of IK(ACh) by diadenosine polyphosphates in guinea-pig atrial myocytes.

1. The effects of diadenosine polyphosphates (APnA, where n = 4-6) were studied on beating frequency of perfused guinea-pig hearts and on muscarinic K+ current (IK(ACh)) and ATP-regulated K+ current (IK(ATP)) in atrial myocytes from guinea-pig hearts using whole-cell voltage clamp. 2. Bradycardia induced by APnA in perfused hearts was completely inhibited by 8-cyclopentyl- 1,3-dipropylxanthine (CPX, 20 microM), a selective antagonist at A1 adenosine receptors, and was augmented by dipyridamole (Dipy), an inhibitor of cellular adenosine (Ado) uptake. 3. Whereas exposure of atrial myocytes to Ado (100 microM) within about 1 s induced a significant whole-cell IK(ACh), APnA up to 1 mM applied for some tens of seconds failed to activate IK(ACh). If present for periods > 2 min, APnA caused inhibition of agonist-evoked IK(ACh) and activation of a weakly inward rectifying K+ current, which was identified as IK(ATP) by its sensitivity to glibenclamide and its current-voltage curve. 4. The actions of extracellular APnA on IK(ACh) and IK(ATP) were mimicked by intracellular loading of compounds via the patch clamp pipette and by intracellular loading of AMP. 5. The results from isolated myocytes exclude APnA acting as A1 agonists. It is suggested that myocytes can take up APnA, which are degraded to AMP. In the presence of ATP, AMP is converted to ADP, a physiological activator of ATP-regulated K+ channels, by adenylate kinase. A similar mechanism resulting in a reduction of the [GTP]/[GDP] ratio might be responsible for inhibition of IK(ACh). 6. In the perfused heart and other multicellular cardiac preparations the actions of APnA are mediated by Ado via A1 receptors. It is suggested that APnA in multicellular cardiac tissue are hydrolysed by an ectohydrolase to yield AMP which is converted to Ado by ectonucleotidases.

Hyperpolarization-activated inward currents contribute to spontaneous electrical activity and CO2/H+ sensitivity of cultivated neurons of fetal rat medulla.

Neurons growing out from cultivated fetal medullary slices that exhibited spontaneous electrical activity after blockade of synaptic transmission were investigated by the patch-clamp technique for their response to decreases in the extracellular pH. Increases in the [H+], induced by increases in pCO2, resulted in a decrease in spike frequency associated with a decrease in the rate of depolarization preceding each action potential. The type of ion channel, contributing to interspike depolarization, and which may therefore be the site of CO2/H+ action, was identified by application of agents that inhibited the hyperpolarization-activated cation, IH, channel (Cs+ and ZD7288). Application of Cs+ and ZD7288 slightly hyperpolarized the cell membrane, decreased the interspike slope and inhibited CO2/H+-induced modulations of spike frequency in one group of CO2-inhibited medullary neurons, suggesting that IH contributes to spontaneous neuronal activity and to CO2/H+-sensitivity. CO2/H+ effects on IH were further confirmed in voltage-clamp experiments. Increasing the bath CO2 from 2% to 9% reduced the IH amplitude, shifted the mean EH from -54 to -60 mV, lengthened the voltage-dependent delay of current activation and increased the time-constants of activation at all potentials studied. It is concluded that depolarizing inward currents through IH channels participate in the gradual ramp-like change in membrane potential which depolarizes the cell up to the threshold of Na+ spike generation. CO2/H+-induced inhibition of IH reduces the contribution of this ion current to the interspike depolarization and accounts for the CO2/H+-induced decrease in spike frequency in one type of CO2/H+-inhibited medullary cells.

Contribution of Ca2+-activated K+ channels to central chemosensitivity in cultivated neurons of fetal rat medulla.

Neurons in fetal rat medullary slices that exhibited spontaneous electrical activity after blockade of synaptic transmission were investigated for their response to decreases in extracellular pH. Increases in [H+] (induced either by fixed acid or increases in PCO2) induced a significant increase in the frequency of action potentials, associated with a membrane depolarization, and/or increases in the slope of the interspike depolarization. In addition, CO2/H+ prolonged the repolarizing phase of action potentials and reduced the afterhyperpolarization, suggesting that K+ channels were the primary site of CO2/H+ action. The type of K+ channel that was modulated by CO2/H+ was identified by application of agents that inhibited Ca2+-activated K+ channels either directly (tetraethylammonium chloride, TEA) or indirectly (Cd2+ ions) by inhibiting Ca2+ influx. CO2/H+ effects on neuronal activity were abolished after application of these blockers. The contribution of Ca2+-activated K+ channels to H+ sensitivity of these neurons was confirmed further in voltage-clamp experiments in which outward rectifying I-V curves were recorded that revealed a zero current potential of -70 mV. CO2/H+ induced a prominent reduction in outward currents and shifted the zero current potential to more positive membrane potentials (mean -63 mV). The CO2/H+-sensitive current reversed at -72 mV and was blocked by external application of TEA. It is concluded that CO2/H+ exerts its stimulatory effects on fetal medullary neurons by inhibition of Ca2+-activated K+ channels, either directly or indirectly, by blocking voltage-dependent Ca2+ channels, which in turn results in a reduction of K+ efflux and in cell depolarization.

CO2-sensitive neurons in organotypic cultures of the fetal rat medulla.

Medullary slices of the fetal rat at gestational day 16 were cultivated (organotypic culture) for up to 20 days and current clamp experiments were performed on outgrowing neurons. CO2-sensitivity was tested by changing the P(CO2) in the bath solution (equilibrating CO2 fraction from 0.02 to 0.09). Two groups of CO2-sensitive neurons were found; one with and the other without intrinsic CO2-chemosensitivity. Neurons with intrinsic CO2-sensitivity maintained their spontaneous activity and chemosensitivity after blockade of synaptic transmission. These neurons exhibited action potentials that were preceeded by a spontaneous interspike depolarization and followed by an afterhyperpolarization (beating neurons). Increasing P(CO2) either decreased (inhibited neurons, n = 55) or increased the spike frequency of these neurons (stimulated neurons, n = 31). The reduced activity of CO2-inhibited neurons was associated with membrane hyperpolarization and/or decreases in the slope of interspike depolarization. In contrast CO2-stimulated neurons were depolarized and the slope of their interspike depolarization was augmented during acidosis. In addition, we demonstrated a strong voltage dependence of CO2-induced effects on membrane potential and spike frequency. Neurons with non-beating activity did not show a spontaneous interspike depolarization and their spike generation and CO2-sensitivity appeared to be entirely produced through synaptic inputs. The CO2-mediated changes in electrical properties of these neurons closely resemble those of various CNS neurons, including respiratory neurons, in whole animal or neonatal brainstem-spinal cord preparations.