Marie-Cécile Wellner-Kienitz

Transfection of a phosphatidyl‐4‐phosphate 5‐kinase gene into rat atrial myocytes removes inhibition of GIRK current by endothelin and α‐adrenergic agonists

GIRK (G protein‐activated inward‐rectifying K+ channel) channels, important regulators of membrane excitability in the heart and in the central nervous, are activated by interaction with βγ subunits from heterotrimeric G proteins upon receptor stimulation. For activation interaction of the channel with phosphatidylinositol 4,5‐bisphosphate (PtIns(4,5)P2) is conditional. Previous studies have provided evidence that in myocytes PtIns(4,5)P2 levels relevant to GIRK channel regulation are under regulatory control of receptors activating phospholipase C. In the present study a phosphatidyl‐4‐phosphate 5‐kinase was expressed in atrial myocytes by transient transfection. This did not affect basal properties of GIRK current activated by acetylcholine via M2 receptors but completely abolished inhibition of guanosine triphosphate‐γ‐S activated current by endothelin‐1 or α‐adrenergic agonists. We conclude that though PtIns(4,5)P2 is conditional for channel gating, its normal level in the membrane is not limiting basal function of GIRK channels. Moreover, our data provide further evidence for a regulation of GIRK channels by α1A receptors and endothelin‐A receptors, endogenously expressed in atrial myocytes, via depletion of PtIns(4,5)P2.

Coupling to Gs and Gq/11 of histamine H2 receptors heterologously expressed in adult rat atrial myocytes

The predominant histamine receptor subtype in the supraventricular and ventricular tissue of various mammalian species is the H2 receptor (H2-R) subtype, which is known to couple to stimulatory G proteins (Gs), i.e. the major effects of this autacoid are an increase in sinus rate and in force of contraction. To investigate histamine effects in H2-R-transfected rat atrial myocytes, endogenous GIRK currents and L-type Ca2+ currents were used as functional assays. In H2-R-transfected myocytes, exposure to His resulted in a reversible augmentation of L-type Ca2+ currents, consistent with the established coupling of this receptor to the Gs-cAMP-PKA signalling pathway. Mammalian K+ channels composed of GIRK (Kir3.x) subunits are directly controlled by interaction with betagamma subunits released from G proteins, which couple to seven-helix receptors. In mock-transfected atrial cardiomyocytes, activation of muscarinic K+ channels (IK(ACh)) was limited to Gi-coupled receptors (M2R, A1R). In H2-R-overexpressing cells, histamine activated IK(ACh) via Gs-derived betagamma subunits since the histamine-induced current was insensitive to pertussis toxin. These data indicate that overexpression of Gs-coupled H2-R results in a loss of target specificity due to an increased agonist-induced release of Gs-derived betagamma subunits. When IK(ACh) was maximally activated by GTP-gamma-S, histamine induced an irreversible inhibition of the inward current in a fraction of H2-R-transfected cells. This inhibition is supposed to be mediated via a G(q/11)-PLC-mediated depletion of PIP2, suggesting a partial coupling of overexpressed H2-R to G(q/11). Dual coupling of H2-Rs to Gs and Gq is demonstrated for the first time in cardiac myocytes. It represents a novel mechanism to augment positive inotropic effects by activating two different signalling pathways via one type of histamine receptor. Activation of the Gs-cAMP-PKA pathway promotes Ca2+ influx through phosphorylation of L-type Ca2+ channels. Simultaneous activation of Gq-signalling pathways might result in phosphoinositide turnover and Ca2+ release from intracellular stores, thereby augmenting H2-induced increases in [Ca2+]i.

Antisense oligonucleotides against receptor kinase GRK2 disrupt target selectivity of β‐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 βγ‐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 Gβγ binding sites, in a fraction of cells, K(ACh) channels can be activated by β‐adrenergic receptors. Sensitivity to β‐agonist is insensitive to pertussis toxin treatment. These findings demonstrate a potential role of Gβγ binding proteins for target selectivity of G‐protein‐coupled receptors

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 μm), 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 μm) 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.

Acute desensitization of GIRK current in rat atrial myocytes is related to K+ current flow

We have investigated the acute desensitization of acetylcholine‐activated GIRK current (IK(ACh)) in cultured adult rat atrial myocytes. Acute desensitization of IK(ACh) is observed as a partial relaxation of current with a half‐time of < 5 s when muscarinic M2 receptors are stimulated by a high concentration (> 2 μmol l−1) of ACh. Under this condition experimental manoeuvres that cause a decrease in the amplitude of IK(ACh), such as partial block of M2 receptors by atropine, intracellular loading with GDP‐β‐S, or exposure to Ba2+, caused a reduction in desensitization. Acute desensitization was also identified as a decrease in current amplitude and a blunting of the response to saturating [ACh] (20 μmol l−1) when the current had been partially activated by a low concentration of ACh or by stimulation of adenosine A1 receptors. A reduction in current analogous to acute desensitization was observed when ATP‐dependent K+ current (IK(ATP)) was activated either by mitochondrial uncoupling using 2,4‐dinitrophenole (DNP) or by the channel opener rilmakalim. Adenovirus‐driven overexpression of Kir2.1, a subunit of constitutively active inwardly rectifying K+ channels, resulted in a large Ba2+‐sensitive background K+ current and a dramatic reduction of ACh‐activated current. Adenovirus‐driven overexpression of GIRK4 (Kir3.4) subunits resulted in an increased agonist‐independent GIRK current paralleled by a reduction in IK(ACh) and removal of the desensitizing component. These data indicate that acute desensitization depends on K+ current flow, independent of the K+ channel species, suggesting that it reflects a reduction in electrochemical driving force rather than a bona fide signalling mechanism. This is supported by the observation that desensitization is paralleled by a significant negative shift in reversal potential of IK(ACh). Since the ACh‐induced hyperpolarization shows comparable desensitization properties as IK(ACh), this novel current‐dependent desensitization is a physiologically relevant process, shaping the time course of parasympathetic bradycardia.

Voltage dependence of ATP‐dependent K+ current in rat cardiac myocytes is affected by IK1 and IK(ACh)

In this study we have investigated the voltage dependence of ATP‐dependent K+ current (IK(ATP)) in atrial and ventricular myocytes from hearts of adult rats and in CHO cells expressing Kir6.2 and SUR2A. The current–voltage relation of 2,4‐dinitrophenole (DNP) ‐induced IK(ATP) in atrial myocytes and expressed current in CHO cells was linear in a voltage range between 0 and −100 mV. In ventricular myocytes, the background current–voltage relation of which is dominated by a large constitutive inward rectifier (IK1), the slope conductance of IK(ATP) was reduced at membrane potentials negative to EK (around −50 mV), resulting in an outwardly rectifying I–V relation. Overexpression of Kir2.1 by adenoviral gene transfer, a subunit contributing to IK1 channels, in atrial myocytes resulted in a large IK1‐like background current. The I–V relation of IK(ATP) in these cells showed a reduced slope conductance negative to EK similar to ventricular myocytes. In atrial myocytes with an increased background inward‐rectifier current through Kir3.1/Kir3.4 channels (IK(ACh)), irreversibly activated by intracellular loading with GTP‐γ‐S, the I–V relation of IK(ATP) showed a reduced slope negative to EK, as in ventricular myocytes and atrial myocytes overexpressing Kir2.1. It is concluded that the voltage dependencies of membrane currents are not only dependent on the molecular composition of the charge‐carrying channel complexes but can be affected by the activity of other ion channel species. We suggest that the interference between inward IK(ATP) and other inward rectifier currents in cardiac myocytes reflects steady‐state changes in K+ driving force due to inward K+ current.

Activation of muscarinic K+ current by β‐adrenergic receptors in cultured atrial myocytes transfected with β1 subunit of heterotrimeric G proteins

Muscarinic K+ channels (IK(ACh)) in native atrial myocytes are activated by βγ subunits of pertussis toxin (Ptx)‐sensitive heterotrimeric G proteins coupled to different receptors. βγ subunits of Ptx‐insensitive Gs, coupled to β‐adrenergic receptors, do not activate native IK(ACh). In atrial myocytes from adult rats transfected with rat brain β1 subunit IK(ACh) can be activated by stimulation of β‐adrenergic receptors using isoprenaline. This effect is insensitive to Ptx. These findings demonstrate for the first time promiscuous (Ptx‐insensitive) coupling of Gsβγ to GIRK channels in their native environment.

Transfection with 5-HT1A receptor gene and antisense directed against muscarinic M2 receptors reveal a mutual influence between Gi/o-coupled receptors in rat atrial myocytes.

A recently described reduction in sensitivity of G protein-activated inward-rectifying K(+) (GIRK) channels to stimulation of muscarinic M(2) receptors (M(2)AChR) in atrial myocytes overexpressing purinergic A(1) receptors (A(1)AdoR) was further investigated by heterologous expression of a 5-HT(1A) receptor (5-HT(1A)R) and by reducing the expression level of endogenous M(2)AChR receptors using antisense. In 5-HT(1A)R-expressing myocytes, in line with previous studies, sizable GIRK currents could be activated by 5-HT. In these cells, the mean current density and activation rate of M(2)AChR-activated current were significantly reduced, supporting the notion that signalling via this receptor is negatively regulated by other G protein-coupled receptors (GPCR) coupling to the same class (G(i/o)) of G proteins. To study if reducing M(2)AChR expression affects sensitivity of GIRK current to stimulation of A(1)AdoR, antisense oligodinucleotides (AsODN) against the M(2)AChR were used. Incubation of myocytes with M(2)AChR-specific AsODN resulted in a significant reduction in mean amplitude and activation rate of ACh-induced currents. This was paralleled by an increase in mean amplitude and activation rate of current activated by stimulation of A(1)AdoR. Plotting amplitudes of 5-HT- or Ado-induced currents from individual manipulated cells against the amplitude of ACh-induced current yielded a positive correlation between these data. Although difficult to interpret in mechanistic terms, this argues against a competition of receptors for a common pool of G(i/o). The mutual interaction between G(i/o)-coupled receptors depends on manipulation of the expression level, since long-term desensitization or down regulation of M(2)AChR by treatment with carbachol did not affect sensitivity of GIRK current to A(1)AdoR stimulation, despite a substantial reduction in amplitude and activation rate of M(2)AChR-activated currents. These data suggest a novel crosstalk between parallel receptors converging on the same class of G proteins.