scholarly journals Heterologous Facilitation of G Protein-Activated K+ Channels by β-Adrenergic Stimulation via Camp-Dependent Protein Kinase

2000 ◽  
Vol 115 (5) ◽  
pp. 547-558 ◽  
Author(s):  
Carmen Müllner ◽  
Dimitry Vorobiov ◽  
Amal Kanti Bera ◽  
Yasuhito Uezono ◽  
Daniel Yakubovich ◽  
...  
Keyword(s):  
G Protein ◽  
Slow Component ◽  
Xenopus Laevis Oocytes ◽  
Adrenergic Stimulation ◽  
Current Increase ◽  
Girk Channels ◽  
Α Subunit ◽  
K Channels ◽  
Adult Rat ◽  
Whole Cell Patch Clamp

To investigate possible effects of adrenergic stimulation on G protein–activated inwardly rectifying K+ channels (GIRK), acetylcholine (ACh)-evoked K+ current, IKACh, was recorded from adult rat atrial cardiomyocytes using the whole cell patch clamp method and a fast perfusion system. The rise time of IKACh was 0.4 ± 0.1 s. When isoproterenol (Iso) was applied simultaneously with ACh, an additional slow component (11.4 ± 3.0 s) appeared, and the amplitude of the elicited IKACh was increased by 22.9 ± 5.4%. Both the slow component of activation and the current increase caused by Iso were abolished by preincubation in 50 μM H89 {N-[2-((p -bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, a potent inhibitor of PKA}. This heterologous facilitation of GIRK current by β-adrenergic stimulation was further studied in Xenopus laevis oocytes coexpressing β2-adrenergic receptors, m2 -receptors, and GIRK1/GIRK4 subunits. Both Iso and ACh elicited GIRK currents in these oocytes. Furthermore, Iso facilitated ACh currents in a way, similar to atrial cells. Cytosolic injection of 30–60 pmol cAMP, but not of Rp-cAMPS (a cAMP analogue that is inhibitory to PKA) mimicked the β2-adrenergic effect. The possibility that the potentiation of GIRK currents was a result of the phosphorylation of the β-adrenergic receptor (β2AR) by PKA was excluded by using a mutant β2AR in which the residues for PKA-mediated modulation were mutated. Overexpression of the α subunit of G proteins (Gαs) led to an increase in basal as well as agonist-induced GIRK1/GIRK4 currents (inhibited by H89). At higher levels of expressed Gαs, GIRK currents were inhibited, presumably due to sequestration of the β/γ subunit dimer of G protein. GIRK1/GIRK5, GIRK1/GIRK2, and homomeric GIRK2 channels were also regulated by cAMP injections. Mutant GIRK1/GIRK4 channels in which the 40 COOH-terminal amino acids (which contain a strong PKA phosphorylation consensus site) were deleted were also modulated by cAMP injections. Hence, the structural determinant responsible is not located within this region. We conclude that, both in atrial myocytes and in Xenopus oocytes, β-adrenergic stimulation potentiates the ACh-evoked GIRK channels via a pathway that involves PKA-catalyzed phosphorylation downstream from β2AR.

Altered myocardial Gs protein and adenylyl cyclase signaling in rats exposed to chronic hypoxia and normoxic recovery

2003 ◽  
Vol 94 (6) ◽  
pp. 2423-2432 ◽  
Author(s):  
Markéta Hrbasová ◽  
Jiri Novotny ◽  
Lucie Hejnová ◽  
František Kolář ◽  
Jan Neckář ◽  
...  
Keyword(s):  
Adenylyl Cyclase ◽  
G Protein ◽  
Contractile Function ◽  
Left Ventricular ◽  
Adaptation To Hypoxia ◽  
Adrenergic Stimulation ◽  
Α Subunit ◽  
Stimulatory G Protein ◽  
Membrane Bound ◽  
The Right

The present work has analyzed the consequences of chronic intermittent high-altitude hypoxia for functioning of the G protein-mediated adenylyl cyclase (AC) signaling system in the right (RV) and left ventricular (LV) myocardium in rats. Adaptation to hypoxia did not appreciably affect the number of β-adrenoceptors and the content of predominantly membrane-bound α-subunit (Gsα) of the stimulatory G protein, but it raised the amount of cytosolic Gsα in RV. The levels of myocardial inhibitory Gα protein were not altered. Activity of AC stimulated by GTP, fluoride, forskolin, or isoprotertenol was reduced by ∼50% in RV from chronically hypoxic rats, and a weaker depression was also found in LV. In addition, hypoxia significantly diminished a functional activity of membrane-bound Gsα in both RV and LV. The RV baseline contractile function was markedly increased in chronically hypoxic animals, and its sensitivity to β-adrenergic stimulation was decreased. Animals recovering from hypoxia for 5 wk still exhibited markedly elevated levels of cytosolic Gsα and significantly lower activity of AC in RV than did age-matched controls, but contractile responsiveness to β-agonists was normal.

Characterization of the Ca2+ current in freshly dissociated crustacean peptidergic neuronal somata

1995 ◽  
Vol 73 (6) ◽  
pp. 2357-2368 ◽  
Author(s):  
J. E. Richmond ◽  
E. Sher ◽  
I. M. Cooke
Keyword(s):  
Slow Component ◽  
Neuronal Somata ◽  
Current Increase ◽  
Inactivation Curve ◽  
Test Pulse ◽  
Whole Cell Patch Clamp ◽  
Recovery From Inactivation ◽  
Dependent Inactivation ◽  
Na Currents ◽  
Voltage Dependent

1. Freshly dissociated neuronal somata of the crab (Cardisoma carnifex) X-organ were studied in the whole cell patch-clamp configuration. To characterize the Ca2+ currents in these somata, recordings were made under conditions designed to suppress K+ and Na+ currents. 2. In 52 mM external Ca2+ the threshold for activation of Ca2+ currents was above -40 mV, with peak amplitudes occurring around +10 to +20 mV. The full component of the current was available for activation at -50 mV because no current increase was observed when the holding potential was increased to -90 mV. These characteristics of the current characterize it as a high-voltage activated (HVA) current. 3. The Ca2+ current was almost completely (60-90%) inactivated within 200 ms at maximal current potentials (+10 to +20 mV). The decay was best described by a double-exponential function with a fast and slow component of inactivation (tau f = 12 ms and tau s = 64 ms). Both Sr2+ and Ba2+ substitutions reduced the rates of inactivation. 4. In double-pulse experiments, plots of variable prepulse potential versus test pulse current produced a U-shaped curve with test pulse currents showing maximal inactivation at potentials that produced maximal Ca2+ influx during the prepulse. Tail currents also displayed a U-shaped inactivation curve. The extent of current-dependent inactivation was sequentially reduced by Sr2+ and Ba2+ substitutions. These data suggest that inactivation in crab somata is predominantly Ca2+ dependent. The remaining inactivation of Ba2+ currents suggests that there is also a component of voltage-dependent inactivation in the somata. 5. Part of the inactivated Ca2+ current could be recovered during short (4-10 ms) hyperpolarizing pulses to -130 mV. The absolute extent of recovery from inactivation was greatest for currents carried by Ca2+ rather than Sr2+ or Ba2+. When voltage-dependent inactivation was dominant (Ba2+ currents), the relative amount of current recovered was greater. The data suggest that hyperpolarizing pulses are more effective in removing voltage-dependent inactivation, but also allow some recovery from Ca(2+)-dependent inactivation. 6. In the crab saline, which contained 24 mM Mg2+, the amplitudes of currents carried by 52 mM Ca2+, Sr2+ and Ba2+ were similar. Removing the Mg2+ from the saline augmented both the Ba2+ and Sr2+ currents relative to the Ca2+ current.(ABSTRACT TRUNCATED AT 400 WORDS)

Human Adrenal Glomerulosa Cells Express K2P and GIRK Potassium Channels that are inhibited by AngII and ACTH

2021 ◽  
Author(s):  
John J. Enyeart ◽  
Judith A. Enyeart
Keyword(s):  
G Protein ◽  
Zona Glomerulosa ◽  
Time Dependent ◽  
Aldosterone Secretion ◽  
Girk Channels ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Girk Channel ◽  
Inwardly Rectifying ◽  
G Protein Coupled

In whole-cell patch clamp recordings, it was discovered that normal human adrenal zona glomerulosa (AZG) cells express members of the three major families of K+ channels. Among these are a two pore (K2P) leak-type and a G-protein-coupled, inwardly-rectifying (GIRK) channel, both inhibited by peptide hormones that stimulate aldosterone secretion. The K2P current displayed properties identifying it as TREK-1 (KCNK2). This outwardly-rectifying current was activated by arachidonic acid and inhibited by angiotensin II (AngII), adrenocorticotrophic hormone (ACTH), and forskolin. The activation and inhibition of TREK-1 was coupled to AZG cell hyperpolarization and depolarization, respectively. A second K2P channel, TASK-1 (KCNK3), was expressed at a lower density in AZG cells. Human AZG cells also express inwardly rectifying K+ current(s) (KIR) that include quasi-instantaneous and time-dependent components. This is the first report demonstrating the presence of KIR in whole cell recordings from AZG cells of any species. The time-dependent current was selectively inhibited by AngII, and ACTH, identifying it as a G protein-coupled (GIRK) channel, most likely KIR3.4 (KCNJ5). The quasi-instantaneous KIR current was not inhibited by AngII or ACTH, and may be a separate non-GIRK current. Finally, AZG cells express a voltage-gated, rapidly inactivating K+ current whose properties identified as KV1.4 (KCNA4), a conclusion confirmed by Northern blot. These findings demonstrate that human AZG cells express K2P and GIRK channels whose inhibition by AngII and ACTH are likely coupled to depolarization-dependent secretion. They further demonstrate that human AZG K+ channels differ fundamentally from the widely adopted rodent models for human aldosterone secretion.

Abstract 17317: Differential Coupling and Regulation of GIRK-Dependent Muscarinic and Adenosine Receptor Signaling in Mouse Sino-Atrial Nodal Cells

Circulation ◽  
2018 ◽  
Vol 138 (Suppl_1) ◽  
Author(s):  
Allison Anderson ◽  
Kevin Wickman
Keyword(s):  
G Protein ◽  
Adenosine Receptor ◽  
Protein Signaling ◽  
Wild Type ◽  
Girk Channels ◽  
Genetic Ablation ◽  
Whole Cell Patch Clamp ◽  
Girk Channel ◽  
Induced Currents ◽  
Patch Clamp Electrophysiology

Introduction and Hypothesis: Parasympathetic slowing of heart rate is largely mediated by muscarinic receptor (M2R) activation of the G protein gated inwardly-rectifying potassium (GIRK) channel on sinoatrial-nodal (SAN) cells and atrial myocytes. The strength of M2R-GIRK signaling is negatively regulated by regulator of G protein signaling 6, RGS6. Genetic ablation of RGS6 in mice gives rise to enhanced M2R-GIRK signaling in SAN cells, resulting in both exaggerated M2R-induced bradycardia and increased susceptibility to pacing-induced atrial fibrillation. Adenosine receptor (A1R) activation, which can provoke atrial arrhythmias in human patients, can also activate GIRK channels, however, the details of A1R-GIRK signaling are poorly understood. Here, we investigated A1R-GIRK signaling in mouse SAN cells and tested the hypothesis that RGS6 negatively regulates A1R-GIRK signaling. Methods and Results: Using whole-cell patch-clamp electrophysiology, we measured the efficacy and potency of M2R- and A1R- GIRK signaling in adult SAN cells from wildtype and Rgs6 -/- mice. We found SAN cells from Rgs6 -/- mice displayed both prolonged channel deactivation kinetics and increased channel sensitivity to CCh-induced currents as compared to wild-type SAN cells. Surprisingly, we found no difference in kinetics or channel sensitivity of A1R-GIRK responses in Rgs6 -/- SAN cells. We did observe, however, a striking, significant increase in the amplitude of the A1R-GIRK response in Rgs6 -/- SAN cells compared to wild-type controls. Furthermore, occlusion studies in wild-type SAN cells suggest that M2R activates nearly all of the GIRK channels present in SAN cells, while A1R activation results in only partial GIRK channel activation. Intriguingly, RGS6 ablation seems to allow a larger proportion of GIRK channels to be activated by A1R. Recordings from mice lacking cardiac GIRK channels confirm that M2R- and A1R- induced currents are GIRK-dependent. Conclusions: Our results suggest that M2R-GIRK and A1R-GIRK are coupled differently within mouse sino-atrial nodal cells, resulting in differential regulation by RGS6.

G protein-mediated suppression of L-type Ca2+ current by interleukin-1 beta in cultured rat ventricular myocytes

1995 ◽  
Vol 268 (2) ◽  
pp. C339-C349 ◽  
Author(s):  
S. Liu ◽  
K. D. Schreur
Keyword(s):  
G Protein ◽  
Voltage Dependence ◽  
Ventricular Myocytes ◽  
Interleukin 1 ◽  
Signal Transduction Pathway ◽  
Interleukin 1 Beta ◽  
Rat Ventricular Myocytes ◽  
Adult Rat ◽  
Whole Cell Patch Clamp ◽  
Steady State Inactivation

The effect and possible signal transduction pathway of interleukin-1 beta (IL-1 beta) on the L-type Ca2+ current (ICa,L) in cultured adult rat ventricular myocytes were examined using whole cell patch-clamp techniques. When myocytes were internally dialyzed with a solution containing GTP, IL-1 beta caused a concentration-dependent decrease in the peak ICa,L (Ba2+ as the charge carrier). IL-1 beta did not significantly alter the voltage dependence of the peak ICa,L nor the steady-state inactivation and activation, but did slightly slow the rate of inactivation. In myocytes dialyzed with solutions without GTP or including guanosine 5'-O-(2-thiodiphosphate) to replace GTP, IL-1 beta had no effect on ICa,L. In contrast, when guanosine 5'-O-(3-thiotriphosphate) was used to replace GTP, the suppression of ICa,L induced by IL-1 beta remained. Preincubation of myocytes with pertussis toxin (PTX), which completely abolished the acetylcholine effect on isoproterenol-stimulated ICa,L, had no effect on the inhibitory action of IL-1 beta on ICa,L. We conclude that in cultured rat ventricular myocytes, IL-1 beta suppresses ICa,L via a PTX-insensitive G protein.

scholarly journals Diterpene Lipo-Alkaloids with Selective Activities on Cardiac K+ Channels

Planta Medica ◽  
2017 ◽  
Vol 83 (17) ◽  
pp. 1321-1328 ◽  
Author(s):  
Tivadar Kiss ◽  
Botond Borcsa ◽  
Péter Orvos ◽  
László Tálosi ◽  
Judit Hohmann ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Ion Channels ◽  
Patch Clamp Technique ◽  
Diterpene Alkaloids ◽  
Girk Channels ◽  
Vast Number ◽  
K Channels ◽  
Whole Cell Patch Clamp ◽  
Girk Channel

Abstract Aconitum diterpene alkaloids are known for their remarkable toxicity, which is due to their effect on ion channels. Activation of voltage-gated Na+ channels is the major cause of their cardiotoxicity, however, influence on K+ channels may also play a role in the overall effect.Here we report the synthesis of a series of lipo-alkaloids, including four new compounds, based on the 14-benzoylaconine structure, which is the core of a vast number of diterpene alkaloids naturally occurring in Aconitum species. The activities of these compounds were measured in vitro on K+ ion channels using the whole-cell patch-clamp technique. Structure-activity analysis was carried out based on the data of 51 compounds (32 genuine diterpene alkaloids, 5 fatty acids, and 14 lipo-alkaloids). Depending on their substitution, these compounds exert different activities on GIRK (G protein-coupled inwardly-rectifying potassium channel) and hERG (human ether-à-go-go-related gene) channels. Fatty acids and diterpene alkaloids show lower activity on the GIRK channel than lipo-alkaloids. Lipo-alkaloids also have less pronounced hERG inhibitory activity compared to the cardiotoxic aconitine. Considering the GIRK/hERG selectivity as an indicator of perspective therapeutic applicability, lipo-alkaloids are significantly more selective than the genuine diterpene alkaloids. 14-Benzoyl-8-O-eicosa-8Z,11Z,14Z-trienoate and 14-benzoyl-8-O-eicosa-11Z,14Z,17Z-trienoate are strong and selective inhibitors of GIRK channels, thus, they are promising subjects for further studies to develop diterpene alkaloid-based antiarrhythmic pharmacons.

Effects of Orexin (Hypocretin) on GIRK Channels

2003 ◽  
Vol 90 (2) ◽  
pp. 693-702 ◽  
Author(s):  
Q. V. Hoang ◽  
D. Bajic ◽  
M. Yanagisawa ◽  
S. Nakajima ◽  
Y. Nakajima
Keyword(s):  
G Protein ◽  
Cellular Mechanisms ◽  
Opioid Agonist ◽  
Initial Current ◽  
Current Increase ◽  
Girk Channels ◽  
Mu Opioid ◽  
Hek Cells ◽  
Orexin Receptors ◽  
Subsequent Decrease

Orexins (hypocretins) are recently discovered excitatory transmitters implicated in arousal and sleep. Yet, their ionic and signal transduction mechanisms have not been fully clarified. Here we show that orexins suppress G-protein–coupled inward rectifier (GIRK) channel activity, and this suppression is likely to lead to neuronal excitation. Cultured neurons from the locus coeruleus (LC) and the nucleus tuberomammillaris (TM) were used, as well as HEK293A cells transfected with GIRK1 and 2, either human orexin receptor type 1 (OX1R) or type 2 (OX2R), mu opioid receptor and GFP cDNAs. In GTPγS-loaded cells, orexin A (OXA, 3 μM) inhibited GIRK currents that had previously been activated by somatostatin (in LC cells), nociceptin (TM cells), or the mu opioid agonist DAMGO (HEK cells). In guanosine triphosphate (GTP)–loaded HEK cells, in which GIRK currents were not preactivated, OXA induced a biphasic response through both types of orexin receptors: an initial current increase and a subsequent decrease to below resting levels. Current–voltage (I–V) relationships revealed that both the OXA-induced and suppressed currents are inwardly rectifying with reversal potentials around E K. The OXA-induced initial current was partially pertussis toxin (PTX) sensitive and partially PTX insensitive, whereas the OXA-suppressed current was PTX insensitive. These data suggest that orexin receptors couple with more than one type of G-protein, including PTX-sensitive (such as Gi/o) and PTX-insensitive (such as Gq/11) G-proteins. The modulation of GIRK channels by orexins may be one of the cellular mechanisms for the regulation of brain nuclei (e.g., LC and TM) that are crucial for arousal, sleep, and appetite.

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