scholarly journals A functional model for G protein activation of the muscarinic K+ channel in guinea pig atrial myocytes. Spectral analysis of the effect of GTP on single-channel kinetics.

1996 ◽  
Vol 108 (6) ◽  
pp. 485-495 ◽  
Author(s):  
Y Hosoya ◽  
M Yamada ◽  
H Ito ◽  
Y Kurachi
Keyword(s):  
Spectral Analysis ◽  
Guinea Pig ◽  
G Protein ◽  
Functional Model ◽  
K Channel ◽  
Channel Activity ◽  
Atrial Myocytes ◽  
Protein Activation ◽  
G Protein Activation ◽  
Active Channels

To elucidate the functional interaction between the active G protein subunit (GK*) and the cardiac muscarinic K+ (KACh) channel, the effect of intracellular GTP on the channel current fluctuation in the presence of 0.5 microM extracellular acetylcholine was examined in inside-out patches from guinea pig atrial myocytes using spectral analysis technique. The power density spectra of current fluctuations induced at various concentrations of GTP ([GTP]) were well fitted by the sum of two Lorentzian functions. Because the channel has one open state, the open-close transitions of the channel gate represented by the spectra could be described as C2<-->C1<-->O. As [GTP] was raised, the channel activity increased in a positive cooperative manner. The powers of the two Lorentzian components concomitantly increased, while the corner frequencies and the ratio of the powers at 0 Hz remained almost constant. This indicates that G protein activation did not affect the gating of each channel but mainly increased the number of functionally active channels in the patch to enhance the channel activity. Regulation of the number of functionally active channels could be described by a slow transition of the channel states, U (unavailable)<-->A (available), which is independent of the gating. The equilibrium of this slow transition was shifted by GTP from U to A. Monod-Wyman-Changeux's allosteric model for the channel state transition(U<-->A) could well describe the positive cooperative increase in the channel availability by GTP, assuming that, in the presence of saturating concentrations of ACh, [GK*] linearly increased as [GTP] was raised in our experimental range. The model indicates that the cardiac KACh channel could be described as a multimer composed of four or more functionally identical subunits, to each of which one GK* binds.

scholarly journals Intrinsic gating properties of a cloned G protein-activated inward rectifier K+ channel.

1995 ◽  
Vol 106 (1) ◽  
pp. 1-23 ◽  
Author(s):  
C A Doupnik ◽  
N F Lim ◽  
P Kofuji ◽  
N Davidson ◽  
H A Lester
Keyword(s):  
G Protein ◽  
K Channel ◽  
Extrinsic Factors ◽  
Time Resolved ◽  
Time Constants ◽  
Protein Activation ◽  
G Protein Activation ◽  
Voltage Dependent ◽  
Current Activation ◽  
Inwardly Rectifying

The voltage-, time-, and K(+)-dependent properties of a G protein-activated inwardly rectifying K+ channel (GIRK1/KGA/Kir3.1) cloned from rat atrium were studied in Xenopus oocytes under two-electrode voltage clamp. During maintained G protein activation and in the presence of high external K+ (VK = 0 mV), voltage jumps from VK to negative membrane potentials activated inward GIRK1 K+ currents with three distinct time-resolved current components. GIRK1 current activation consisted of an instantaneous component that was followed by two components with time constants tau f approximately 50 ms and tau s approximately 400 ms. These activation time constants were weakly voltage dependent, increasing approximately twofold with maximal hyperpolarization from VK. Voltage-dependent GIRK1 availability, revealed by tail currents at -80 mV after long prepulses, was greatest at potentials negative to VK and declined to a plateau of approximately half the maximal level at positive voltages. Voltage-dependent GIRK1 availability shifted with VK and was half maximal at VK -20 mV; the equivalent gating charge was approximately 1.6 e-. The voltage-dependent gating parameters of GIRK1 did not significantly differ for G protein activation by three heterologously expressed signaling pathways: m2 muscarinic receptors, serotonin 1A receptors, or G protein beta 1 gamma 2 subunits. Voltage dependence was also unaffected by agonist concentration. These results indicate that the voltage-dependent gating properties of GIRK1 are not due to extrinsic factors such as agonist-receptor interactions and G protein-channel coupling, but instead are analogous to the intrinsic gating behaviors of other inwardly rectifying K+ channels.

Structure, G Protein Activation, and Functional Relevance of the Cardiac G Protein-Gated K+ Channel, IKACh

1999 ◽  
Vol 868 (1 MOLECULAR AND) ◽  
pp. 386-398 ◽  
Author(s):  
KEVIN WICKMAN ◽  
GRIGORY KRAPIVINSKY ◽  
SHAWN COREY ◽  
MATT KENNEDY ◽  
JAN NEMEC ◽  
...  
Keyword(s):  
G Protein ◽  
K Channel ◽  
Protein Activation ◽  
G Protein Activation ◽  
Functional Relevance

Muscarine-gated K+ channel: subunit stoichiometry and structural domains essential for G protein stimulation

1996 ◽  
Vol 271 (1) ◽  
pp. H379-H385 ◽  
Author(s):  
S. J. Tucker ◽  
M. Pessia ◽  
J. P. Adelman
Keyword(s):  
G Protein ◽  
Xenopus Oocytes ◽  
K Channel ◽  
Channel Activity ◽  
Atrial Myocytes ◽  
Structural Domains ◽  
Maximal Stimulation ◽  
Subunit Stoichiometry ◽  
K Current ◽  
Inwardly Rectifying

Coexpression in Xenopus oocytes of the cloned cardiac inward rectifier subunits Kir 3.1 and Kir 3.4 results in G protein-stimulated channel activity closely resembling the muscarinic channel underlying the inwardly rectifying K+ current in atrial myocytes. To determine the stoichiometry and relative subunit positions within the channel, Kir 3.1 and Kir 3.4 were coexpressed in varying ratios with cloned G beta 1 gamma 2 subunits and also as tandemly linked tetramers with different relative subunit positions. The results reveal that the most efficient channel comprises two subunits of each type in an alternating array within the tetramer. To localize regions important for subunit coassembly and G protein sensitivity, chimeric subunits containing domains from either Kir 3.1, Kir 3.4, or the G protein-insensitive subunit Kir 4.1 were expressed. The results demonstrate that the transmembrane domains dictate the potentiation of the coassembled channels and that, although the NH4- or COOH-termini of both subunits alone can confer G protein sensitivity, both termini are required for maximal stimulation by G beta 1 gamma 2.

scholarly journals On the mechanism of basal and agonist-induced activation of the G protein-gated muscarinic K+ channel in atrial myocytes of guinea pig heart.

1991 ◽  
Vol 98 (3) ◽  
pp. 517-533 ◽  
Author(s):  
H Ito ◽  
T Sugimoto ◽  
I Kobayashi ◽  
K Takahashi ◽  
T Katada ◽  
...  
Keyword(s):  
Guinea Pig ◽  
G Protein ◽  
K Channel ◽  
Dependent Manner ◽  
Atrial Myocytes ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Concentration Dependent Manner ◽  
Guinea Pig Heart ◽  
Gamma S

Using the patch clamp technique, we examined the agonist-free, basal interaction between the muscarinic acetylcholine (m-ACh) receptor and the G protein (GK)-gated muscarinic K+ channel (IK.ACh), and the modification of this interaction by ACh binding to the receptor in single atrial myocytes of guinea pig heart. In the whole cell clamp mode, guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma S) gradually increased the IK.ACh current in the absence of agonists (e.g., acetylcholine). This increase was inhibited in cells that were pretreated with islet-activating protein (IAP, pertussis toxin) or N-ethylmaleimide (NEM). In inside-out patches, even in the absence of agonists, intracellular GTP caused openings of IK.ACh in a concentration-dependent manner in approximately 80% of the patches. Channel activation by GTP in the absence of agonist was much less than that caused by GTP-gamma S. The agonist-independent, GTP-induced activation of IK.ACh was inhibited by the A promoter of IAP (with nicotinamide adenine dinucleotide) or NEM. As the ACh concentration was increased, the GTP-induced maximal open probability of IK.ACh was increased and the GTP concentration for the half-maximal activation of IK.ACh was decreased. Intracellular GDP inhibited the GTP-induced openings of IK.ACh in a concentration-dependent fashion. The half-inhibition of IK.ACh openings occurred at a much lower concentration of GDP in the absence of agonists than in the presence of ACh. From these results, we concluded (a) that the interaction between the m-ACh receptor and GK is essential for basal stimulation of IK.ACh, and (b) that ACh binding to the receptor accelerates the turnover of GK and increases GK's affinity to GTP analogues over GDP.

scholarly journals Na+ activation of the muscarinic K+ channel by a G-protein-independent mechanism.

1996 ◽  
Vol 108 (5) ◽  
pp. 381-391 ◽  
Author(s):  
J L Sui ◽  
K W Chan ◽  
D E Logothetis
Keyword(s):  
G Protein ◽  
Cardiac Glycosides ◽  
Time Course ◽  
K Channel ◽  
Protein Activation ◽  
Atrial Cells ◽  
Gating Mechanism ◽  
Open Time ◽  
Heterologous System ◽  
G Protein Activation

Muscarinic potassium channels (KACh) are composed of two subunits, GIRK1 and GIRK4 (or CIR), and are directly gated by G proteins. We have identified a novel gating mechanism of KACh, independent of G-protein activation. This mechanism involved functional modification of KACh which required hydrolysis of physiological levels of intracellular ATP and was manifested by an increase in the channel mean open time. The ATP-modified channels could in turn be gated by intracellular Na+, starting at approximately 3 mM with an EC50 of approximately 40 mM. The Na(+)-gating of KACh was operative both in native atrial cells and in a heterologous system expressing recombinant channel subunits. Block of the Na+/K+ pump (e.g., by cardiac glycosides) caused significant activation of KACh in atrial cells, with a time course similar to that of Na+ accumulation and in a manner indistinguishable from that of Na(+)-mediated activation of the channel, suggesting that cardiac glycosides activated KACh by increasing intracellular Na+ levels. These results demonstrate for the first time a direct effect of cardiac glycosides on atrial myocytes involving ion channels which are critical in the regulation of cardiac rhythm.

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