Andreas Rinne

Voltage regulates adrenergic receptor function.

The present study demonstrates that agonist-mediated activation of α2A adrenergic receptors (α2AAR) is voltage-dependent. By resolving the kinetics of conformational changes of α2AAR at defined membrane potentials, we show that negative membrane potentials in the physiological range promote agonist-mediated activation of α2AAR. We discovered that the conformational change of α2AAR by voltage is independent from receptor-G protein docking and regulates receptor signaling, including β-arrestin binding, activation of G proteins, and G protein-activated inwardly rectifying K+ currents. Comparison of the dynamics of voltage-dependence of clonidine- vs. norepinephrine-activated receptors uncovers interesting mechanistic insights. For norepinephrine, the time course of voltage-dependent deactivation reflected the deactivation kinetics of the receptor after agonist withdrawal and was strongly attenuated at saturating concentrations. In contrast, clonidine-activated α2AAR were switched by voltage even under fully saturating concentrations, and the kinetics of this switch was notably faster than dissociation of clonidine from α2AAR, indicating voltage-dependent regulation of the efficacy. We conclude that adrenergic receptors exhibit a unique, agonist-dependent mechanism of voltage-sensitivity that modulates downstream receptor signaling.

G Protein-Activated (GIRK) Current in Rat Ventricular Myocytes is Masked by Constitutive Inward Rectifier Current (I K1 )

Inwardly-rectifying K⁺ channel subunits are not homogenously expressed in different cardiac tissues. In ventricular myocytes (VM) the background current-voltage relation is dominated by I_K1, carried by channels composed of Kir2.x subunits, which is less important in atrial myocytes (AM). On the other hand in AM a large G protein gated current carried by Kir3.1/3.4 complexes can be activated by stimulation of muscarinic M₂ receptors (I_K(ACh)), which is assumed to be marginal in VM. Recent evidence suggests that total current carried by cardiac inward-rectifiers (I_K(ATP), I_K(ACh), I_K1) in both, AM and VM is limited, due to K⁺ accumulation/depletion. This lead us to hypothesize that in conventional whole celI recordings I_K(ACh) in VM is underestimated as a consequence of constitutive I_K1. In that case a reduction in density of I_K1 should be paralleled by an increase in density of I_K(ACh). Three different experimental strategies have been used to test for this hypothesis: (i) Adenovirus-driven expression of a dominant-negative mutant of Kir2.1, one of the subunits supposed to form I_K1 channels, in VM caused a reduction in I_K1-density by about 80 %. In those cells I_K(ACh) was increased about 4 fold. (ii) A comparable increase in I_K(ACh) was observed upon reduction of I_K1 caused by adenovirus-mediated RNA interference.(iii) Ba²⁺ in a concentration of 2 µM blocks I_K1 in VM by about 60 % without affecting atrial I_K(ACh). The reduction in I_K1 by 2 µM Ba²⁺ is paralleled by a reversible increase in I_K(ACh) by about 100%. These data demonstrate that the increase in K⁺ conductance underlying ventricular I_K(ACh) is largely underestimated, suggesting that it might be of greater physiological relevance than previously thought.

Generation of a constitutive Na+‐dependent inward‐rectifier current in rat adult atrial myocytes by overexpression of Kir3.4

Apart from gating by interaction with βγ subunits from heterotrimeric G proteins upon stimulation of appropriate receptors, Kir.3 channels have been shown to be gated by intracellular Na+. However, no information is available on how Na+‐dependent gating affects endogenous Kir3.1/Kir3.4 channels in mammalian atrial myocytes. We therefore studied how loading of adult atrial myocytes from rat hearts via the patch pipette filling solution with different concentrations of Na+ ([Na+]pip) affects Kir3 current. Surprisingly, in a range between 0 and 60 mm, Na+ neither had an effect on basal inward‐rectifier current nor on the current activated by acetylcholine. Overexpression of Kir3.4 in adult atrial myocytes forced by adenoviral gene transfer results in formation of functional homomeric channels that interact with βγ subunits upon activation of endogenous muscarinic receptors. These channels are activated at [Na+]pip≥ 15 mm, resulting in a receptor‐independent basal inward rectifier current (Ibir). Ibir was neither affected by pertussis toxin nor by GDP‐β‐S, suggesting G‐protein‐independent activation. PIP2 depletion via endogenous PLC‐coupled α1 adrenergic receptors causes inhibition of endogenous Kir3.1/3.4 channel currents by about 75%. In contrast, inhibition of Na+‐activated Ibir amounts to < 20%. The effect of the Kir3 channel blocker tertiapin‐Q can be described using an IC50 of 12 nm (endogenous IK(ACh)) and 0.61 nm (Ibir). These data clearly identify Ibir as a homotetrameric Kir3.4 channel current with novel properties of regulation and pharmacology. Ibir shares some properties with a basal current recently described in atrial myocytes from an animal model of atrial fibrillation (AF) and AF patients.

Early subcellular Ca2+ remodelling and increased propensity for Ca2+ alternans in left atrial myocytes from hypertensive rats

AIMS Hypertension is a major risk factor for atrial fibrillation. We hypothesized that arterial hypertension would alter atrial myocyte calcium (Ca2+) handling and that these alterations would serve to trigger atrial tachyarrhythmias. METHODS AND RESULTS Left atria or left atrial (LA) myocytes were isolated from spontaneously hypertensive rats (SHR) or normotensive Wistar-Kyoto (WKY) controls. Early after the onset of hypertension, at 3 months of age, there were no differences in Ca2+ transients (CaTs) or expression and phosphorylation of Ca2+ handling proteins between SHR and WKY. At 7 months of age, when left ventricular (LV) hypertrophy had progressed and markers of fibrosis were increased in left atrium, CaTs (at 1 Hz stimulation) were still unchanged. Subcellular alterations in Ca2+ handling were observed, however, in SHR atrial myocytes including (i) reduced expression of the α1C subunit of and reduced Ca2+ influx through L-type Ca2+ channels, (ii) reduced expression of ryanodine receptors with increased phosphorylation at Ser2808, (iii) decreased activity of the Na+ / Ca2+ exchanger (at unaltered intracellular Na+ concentration), and (iv) increased SR Ca2+ load with reduced fractional release. These changes were associated with an increased propensity of SHR atrial myocytes to develop frequency-dependent, arrhythmogenic Ca2+ alternans. CONCLUSIONS In SHR, hypertension induces early subcellular LA myocyte Ca2+ remodelling during compensated LV hypertrophy. In basal conditions, atrial myocyte CaTs are not changed. At increased stimulation frequency, however, SHR atrial myocytes become more prone to arrhythmogenic Ca2+ alternans, suggesting a link between hypertension, atrial Ca2+ homeostasis, and development of atrial tachyarrhythmias.

The mode of agonist binding to a G protein–coupled receptor switches the effect that voltage changes have on signaling

Whether changes in membrane potential enhance or inhibit GPCR signaling depends on the binding position of the ligand. Agonist control of GPCR voltage sensitivity Most G protein–coupled receptors (GPCRs) are activated by ligand binding, but some are also affected by changes in plasma membrane potential, which can either enhance or inhibit GPCR-mediated signaling. Through FRET-based experiments in single cells, Rinne et al. found that depolarization enhanced signaling by the M3 muscarinic acetylcholine receptor when the receptor was bound to the agonists choline or pilocarpine; however, depolarization attenuated M3 receptor signaling when either carbachol or acetylcholine was bound. Molecular docking simulations showed that each group of agonists adopted a distinct binding position. Mutation of a critical residue in the binding pocket changed the binding position of carbachol and switched the response of the carbachol-bound receptor so that signaling was enhanced by membrane depolarization. Together, these data suggest that the binding mode of the agonist determines whether membrane potential changes will enhance or attenuate GPCR signals. These results provide a potential molecular mechanism for drugs that are agonists of specific GPCRs, yet have distinct effects. Signaling by many heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) is either enhanced or attenuated by changes in plasma membrane potential. To identify structural correlates of the voltage sensitivity of GPCR signaling, we chose muscarinic acetylcholine receptors (the M1, M3, and M5 isoforms) as a model system. We combined molecular docking analysis with Förster resonance energy transfer (FRET)–based assays that monitored receptor activity under voltage clamp conditions. When human embryonic kidney (HEK) 293 cells expressing the individual receptors were stimulated with the agonist carbachol, membrane depolarization enhanced signaling by the M1 receptor but attenuated signaling by the M3 and M5 receptors. Furthermore, whether membrane depolarization enhanced or inhibited receptor signaling depended on the type of agonist. Membrane depolarization attenuated M3 receptor signaling when the receptor was bound to carbachol or acetylcholine, whereas depolarization enhanced signaling when the receptor was bound to either choline or pilocarpine. Docking calculations predicted that there were two distinct binding modes for these ligands, which were associated with the effect of depolarization on receptor function. From these calculations, we identified a residue in the M3 receptor that, when mutated, would alter the binding mode of carbachol to resemble that of pilocarpine in silico. Introduction of this mutated M3 receptor into cells confirmed that the membrane depolarization enhanced, rather than attenuated, signaling by the carbachol-bound receptor. Together, these data suggest that the directionality of the voltage sensitivity of GPCR signaling is defined by the specific binding mode of each ligand to the receptor.

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.

Dynamics of Gαi1 interaction with type 5 adenylate cyclase reveal the molecular basis for high sensitivity of Gi-mediated inhibition of cAMP production

Many physiological and pathophysiological processes are regulated by cAMP. Different therapies directly or indirectly influence the cellular concentration of this second messenger. A wide variety of receptors either activates or inhibits adenylate cyclases in order to induce proper physiological responses. A key event in this signalling system is the direct and dynamic interaction of Gαi1 subunits with adenylate cyclases. We established a FRET-based assay between G-protein subunits and AC5 (type 5 adenylate cyclase) and monitored receptor-stimulated interactions between Gαi1 and AC5 in single intact cells with high temporal resolution. We observed that FRET between Gαi1 and AC5 developed at much lower concentration of agonist compared with the overall Gi-protein activity resulting in a left-shift of the concentration-response curve by approximately one order of magnitude. Furthermore, Gi1-protein-mediated attenuation of AC5-dependent increases in cAMP occurred at comparable low concentrations of agonist. On analysing the dynamics we found the dissociation of the Gαi1 subunits and AC5 to occur significantly slower than the G-protein deactivation and to be insensitive to RGS4 (regulator of G-protein signalling type 4) expression. This led us to the conclusion that AC5, by binding active Gαi1, interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation.

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.

Adenovirus-Mediated Delivery of Short Hairpin RNA (shRNA) Mediates Efficient Gene Silencing in Terminally Differentiated Cardiac Myocytes

RNA interference (RNAi) represents the most frequently utilized technique to analyze proteins by loss of function assays. Protein synthesis is impaired by sequence-specific degradation of mRNA, which is triggered by short (19-28 nt) silencing RNAs (siRNA). Efficient gene silencing using RNAi has been demonstrated in numerous cell lines and primary cultured cells. Incorporation of siRNA into terminally differentiated mammalian cells, such as adult cardiac myocytes is limited by their resistance to standard transfection protocols. Viral delivery of short-hairpin RNA (shRNA) overcomes these limitations and allows efficient gene silencing in these cells. This chapter describes the generation and characterization of recombinant siRNA-encoding adenoviruses and their application to adult cardiac myocytes, which represent a standard experimental model in research related to cardiac physiology and pathophysiology. Feasibility of this approach is demonstrated by effective ablation (>80%) of both, a transgene encoding for eGFP and the endogenous muscarinic M(2) acetylcholine receptor.

Membrane Potential Controls the Efficacy of Catecholamine-induced β1-Adrenoceptor Activity

Background: The activity of several Gq- and Gi-coupled receptors is modulated by the membrane potential. Results: Voltage modulates catecholamine-mediated activation of Gs-coupled β1- and β2-adrenoceptors. Conclusion: Voltage-dependence of β1-AR is due to alterations in the efficacy of catecholamines. Significance: By modulating catecholamine efficacy on β1-ARs, voltage can modify receptor activity on a very fast time scale. G protein-coupled receptors (GPCRs) are membrane-located proteins and, therefore, are exposed to changes in membrane potential (VM) in excitable tissues. These changes have been shown to alter receptor activation of certain Gi-and Gq-coupled GPCRs. By means of a combination of whole-cell patch-clamp and Förster resonance energy transfer (FRET) in single cells, we demonstrate that the activation of the Gs-coupled β1-adrenoreceptor (β1-AR) by the catecholamines isoprenaline (Iso) and adrenaline (Adr) is regulated by VM. This voltage-dependence is also transmitted to G protein and arrestin 3 signaling. Voltage-dependence of β2-AR activation, however, was weak compared with β1-AR voltage-dependence. Drug efficacy is a major target of β1-AR voltage-dependence as depolarization attenuated receptor activation, even under saturating concentrations of agonists, with significantly faster kinetics than the deactivation upon agonist withdrawal. Also the efficacy of the endogenous full agonist adrenaline was reduced by depolarization. This is a unique finding since reports of natural full agonists at other voltage-dependent GPCRs only show alterations in affinity during depolarization. Based on a Boltzmann function fit to the relationship of VM and receptor-arrestin 3 interaction we determined the voltage-dependence with highest sensitivity in the physiological range of membrane potential. Our data suggest that under physiological conditions voltage regulates the activity of agonist-occupied β1-adrenoceptors on a very fast time scale.