Coupling to Gs and G(q/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.

Transfection of a phosphatidyl-4-phosphate 5-kinase gene into rat atrial myocytes removes inhibition of GIRK current by endothelin and alpha-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 betagamma subunits from heterotrimeric G proteins upon receptor stimulation. For activation interaction of the channel with phosphatidylinositol 4,5-bisphosphate (PtIns(4,5)P(2)) is conditional. Previous studies have provided evidence that in myocytes PtIns(4,5)P(2) 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 M(2) receptors but completely abolished inhibition of guanosine triphosphate-gamma-S activated current by endothelin-1 or alpha-adrenergic agonists. We conclude that though PtIns(4,5)P(2) 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 alpha(1A) receptors and endothelin-A receptors, endogenously expressed in atrial myocytes, via depletion of PtIns(4,5)P(2).

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.

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 (I(K(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 I(K(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 (I(K1)), the slope conductance of I(K(ATP)) was reduced at membrane potentials negative to E(K) (around -50 mV), resulting in an outwardly rectifying I-V relation. Overexpression of Kir2.1 by adenoviral gene transfer, a subunit contributing to I(K1) channels, in atrial myocytes resulted in a large I(K1)-like background current. The I-V relation of I(K(ATP)) in these cells showed a reduced slope conductance negative to E(K) similar to ventricular myocytes. In atrial myocytes with an increased background inward-rectifier current through Kir3.1/Kir3.4 channels (I(K(ACh))), irreversibly activated by intracellular loading with GTP-gamma-S, the I-V relation of I(K(ATP)) showed a reduced slope negative to E(K), 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 I(K(ATP)) and other inward rectifier currents in cardiac myocytes reflects steady-state changes in K+ driving force due to inward K+ current.

G protein-independent inhibition of GIRK current by adenosine in rat atrial myocytes overexpressing A1 receptors after adenovirus-mediated gene transfer.

G protein-activated inwardly rectifying K+ (GIRK) channels, important regulators of membrane excitability in the heart and central nervous system, are activated by interaction with betagamma subunits from heterotrimeric G proteins upon receptor stimulation. In atrial myocytes various endogenous receptors couple to GIRK channels, including the canonical muscarinic M2 receptor (M2AChR) and the A1 adenosine receptor (A1AdoR). Saturating stimulation of A1AdoR in atrial myocytes activates only a fraction of the GIRK current that is activated via M2AChR, which reflects a lower density of A1AdoR. In the present study A1AdoR were overexpressed by means of adenovirus-mediated gene transfer using green fluorescent protein (GFP) as the reporter. Confirmatory to a previous study, this resulted in an increased sensitivity of macroscopic GIRK current (ACh-activated K+ current (IK(ACh))) to stimulation by Ado. However, in the majority of GFP-positive myocytes, exposure to Ado at concentrations > or =1 microM resulted in activation of IK(ACh) followed by a rapid inhibition. In those cells a rebound activation of current was recorded upon washout of Ado. The inhibitory component could be recorded in isolation when IK(ACh) was activated by M2AChR-stimulation and brief pulses of Ado were superimposed. In myocytes loaded with GTP-gamma-S, IK(ACh), irreversibly activated by brief exposure to agonist, was still reversibly inhibited by Ado, suggesting that inhibition is independent of G protein cycling. In myocytes co-transfected with adenoviral vectors encoding A1AdoR and GIRK4 subunit, no inhibition of GIRK current by Ado was observed. As acute desensitization of atrial GIRK current, which is reminiscent of the inhibition described here, has been shown to be absent in myocytes overexpressing GIRK4, this suggests that acute desensitization and the novel inhibition might share a common pathway whose target is the GIRK channel complex or its GIRK1 subunit.

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 (I(K(ACh))) in cultured adult rat atrial myocytes. Acute desensitization of I(K(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 micromol l(-1)) of ACh. Under this condition experimental manoeuvres that cause a decrease in the amplitude of I(K(ACh)), such as partial block of M2 receptors by atropine, intracellular loading with GDP-beta-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 micromol 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 (I(K(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 I(K(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 I(K(ACh)). Since the ACh-induced hyperpolarization shows comparable desensitization properties as I(K(ACh)), this novel current-dependent desensitization is a physiologically relevant process, shaping the time course of parasympathetic bradycardia.