scholarly journals GTP-dependent regulation of myometrial KCa channels incorporated into lipid bilayers.

1990 ◽  
Vol 96 (2) ◽  
pp. 373-394 ◽  
Author(s):  
L Toro ◽  
J Ramos-Franco ◽  
E Stefani
Keyword(s):  
G Protein ◽  
Lipid Bilayers ◽  
Adrenergic Receptors ◽  
Single Channel ◽  
Channel Activity ◽  
Beta Adrenergic Receptors ◽  
Open Probability ◽  
Beta Adrenergic ◽  
Kca Channels ◽  
Gamma S

The regulation of calcium-activated K (KCa) channels by a G protein-mediated mechanism was studied. KCa channels were reconstituted in planar lipid bilayers by fusion of membrane vesicles from rat or pig myometrium. The regulatory process was studied by exploring the actions of GTP and GTP gamma S on single channel activity. KCa channels had a conductance of 260 +/- 6 pS (n = 25, +/- SE, 250/50 mM KCl gradient) and were voltage dependent. The open probability (Po) vs. voltage relationships were well fit by a Boltzmann distribution. The slope factor (11 mV) was insensitive to internal Ca2+. The half activation potential (V1/2) was shifted -70 mV by raising internal Ca2+ from pCa 6.2 to pCa 4. Addition of GTP or GTP gamma S activated channel activity only in the presence of Mg2+, a characteristic typical of G protein-mediated mechanisms. The Po increased from 0.18 +/- 0.08 to 0.49 +/- 0.07 (n = 7, 0 mV, pCa 6 to 6.8). The channel was also activated (Po increased from 0.03 to 0.37) in the presence of AMP-PNP, a nonphosphorylating ATP analogue, suggesting a direct G protein gating of KCa channels. Upon nucleotide activation, mean open time increased by a factor of 2.7 +/- 0.7 and mean closed time decreased by 0.2 +/- 0.07 of their initial values (n = 6). Norepinephrine (NE) or isoproterenol potentiated the GTP-mediated activation of KCa channels (Po increased from 0.17 +/- 0.06 to 0.35 +/- 0.07, n = 10). These results suggest that myometrium possesses beta-adrenergic receptors coupled to a GTP-dependent protein that can directly gate KCa channels. Furthermore, KCa channels, beta-adrenergic receptors, and G proteins can be reconstituted in lipid bilayers as a stable, functionally coupled, molecular complex.

U46619, a thromboxane A2 agonist, inhibits KCa channel activity from pig coronary artery

1992 ◽  
Vol 262 (3) ◽  
pp. C708-C713 ◽  
Author(s):  
F. S. Scornik ◽  
L. Toro
Keyword(s):  
Smooth Muscle ◽  
Coronary Artery ◽  
Lipid Bilayers ◽  
Thromboxane A2 ◽  
Inhibitory Effect ◽  
Channel Activity ◽  
Open Probability ◽  
Control Value ◽  
Kca Channels ◽  
Internal Side

Thromboxane A2 (TxA2) is a potent vasoconstrictor derived from the metabolism of arachidonic acid. Because potassium channels are involved in the contraction of vascular smooth muscle, their blockade could contribute to the TxA2-induced contraction. To test this possibility, we studied the effect of the TxA2 stable analogue U46619 on calcium-activated potassium (KCa) channels from coronary artery reconstituted into lipid bilayers. Addition of U46619 (50-150 nM) to the external but not to the internal side of the channel decreased the channel open probability (Po) between 15 and 80% of the control value. The inhibitory effect of U46619 affected both the open and closed states of the channel and could be reversed by internal calcium. Thromboxane B2, the inactive hydrolysis derivative of TxA2, did not affect channel activity. SQ 29548, a TxA2 receptor antagonist, was able to prevent the inhibition by U46619. Furthermore, SQ 29548 added after U46619 could restore channel activity to near control values. These results suggest that TxA2 could be a regulatory factor of KCa channels from coronary smooth muscle and that this regulation could be related to its action as a vasoconstrictor.

Ethanol enhances caffeine-induced Ca2+-release channel activation in skeletal muscle sarcoplasmic reticulum

1997 ◽  
Vol 272 (2) ◽  
pp. C622-C627 ◽  
Author(s):  
T. Oba ◽  
M. Koshita ◽  
M. Yamaguchi
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Lipid Bilayers ◽  
Skeletal Muscles ◽  
Single Channel ◽  
Channel Activity ◽  
Electrical Parameters ◽  
Open Probability ◽  
Release Channel ◽  
Channel Current ◽  
Open Time

When sarcoplasmic reticulum (SR) vesicles prepared from frog skeletal muscles were actively loaded with Ca2+, pretreatment of the SR with 2.2 mM (0.01%) ethanol for 30 s significantly potentiated 5 mM caffeine-induced release of Ca2+ from 16.7 +/- 3.7 nmol/mg protein in control without ethanol to 28.0 +/- 2.6 nmol/mg (P < 0.05, n = 5). Ethanol alone caused no release of Ca2+ from the SR. Exposure of the Ca2+-release channel, incorporated into planar lipid bilayers, to 2 mM caffeine significantly increased open probability (Po) and mean open time, but unitary conductance was not affected. Ethanol (2.2 mM) enhanced caffeine-induced Ca2+-release channel activity, with Po reaching 3.02-fold and mean open time 2.85-fold the values in the absence of ethanol. However, ethanol alone did not affect electrical parameters of single-channel current, over a concentration range of 2.2 mM (0.01%) to 217 mM (1%). The synergistic action of ethanol and caffeine on the channel activity could be attributable to enhancement of caffeine-induced release of Ca2+ from the SR vesicles in the presence of ethanol.

Acetylcholine-sensitive potassium channels in human atrial myocytes

1990 ◽  
Vol 259 (6) ◽  
pp. H1730-H1735 ◽  
Author(s):  
R. Sato ◽  
I. Hisatome ◽  
J. A. Wasserstrom ◽  
C. E. Arentzen ◽  
D. H. Singer
Keyword(s):  
Single Channel ◽  
K Channel ◽  
Channel Activity ◽  
Atrial Myocytes ◽  
Open Probability ◽  
Human Atrial Myocytes ◽  
Open Time ◽  
Patch Pipette ◽  
Single Channel Activity ◽  
Gamma S

Single channel recording techniques were used to study acetylcholine (ACh)-sensitive K+ channel activity in human atrial myocytes isolated from specimens obtained during corrective cardiac surgery. Under conditions of cell-attached patch, the presence of ACh in the patch pipette activated K+ channels. Single channel activity occurred in periodic bursts. The channels exhibited a slope conductance of 46 +/- 2 pS inwardly (means +/- SD, n = 4). During a burst, both open and closed time histograms were fitted by a single exponential curve, suggesting the existence of one open and one closed state during a burst. Open probability increased directly with ACh concentration without affecting open time. The channel could be activated by GTP and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) (in the presence and absence of ACh in the pipette, respectively). Slope conductance, the response to GTP and GTP gamma S, and the independence of activation from Ca2+ were similar to those for other species. In contrast, sensitivity to ACh appeared diminished compared with frog atrial myocytes.

scholarly journals The effects of free [Ca2+] on the cytosolic face of the inositol (1,4,5)-trisphosphate receptor at the single channel level

1998 ◽  
Vol 330 (1) ◽  
pp. 559-564 ◽  
Author(s):  
C. Edwin THROWER ◽  
J. A. Edward LEA ◽  
P. Alan DAWSON
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Planar Lipid Bilayers ◽  
Channel Activity ◽  
Open Probability ◽  
Channel Current ◽  
Single Channel Current ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor ◽  
Artificial Bilayers

Cytosolic free Ca2+ has been shown to have both activating and inhibitory effects upon the inositol (1,4,5) trisphosphate receptor (InsP3R) during intracellular Ca2+ release. The effects of cytosolic free Ca2+ on the InsP3R have already been monitored using cerebellar microsomes (containing InsP3R) incorporated into planar lipid bilayers [Bezprozvanny, Watras and Ehrlich (1991) Nature (London) 351, 751-754]. In these experiments the open probability of the channel exhibited a ‘bell-shaped Ca2+ dependence’. However, this has only been seen when the receptor is in the presence of its native membrane (e.g. microsomal vesicles). Using solubilized, purified InsP3R incorporated into planar lipid bilayers using the ‘tip-dip’ technique, investigations were carried out to see if the same effect was seen in the absence of the native membrane. Channel activity was observed in the presence of 4 μM InsP3 and 200 nM free Ca2+. Mean single channel current was 2.69 pA and more than one population of lifetimes was observed. Two populations had mean open times of approx. 9 and 97 ms. Upon increasing the free [Ca2+] to 2 μM, the mean single channel current decreased slightly to 2.39 pA, and the lifetimes increased to 30 and 230 ms. Elevation of free [Ca2+] to 4 μM resulted in a further decrease in mean single channel current to 1.97 pA as well as a decrease in lifetime to approx. 8 and 194 ms. At 10 μM free [Ca2+] no channel activity was observed. Thus, with purified receptor in artificial bilayers, free [Ca2+] on the cytosolic face of the receptor has major effects on channel behaviour, particularly on channel closure, although inhibition of channel activity is not seen until very high free [Ca2+] is reached.

A calcium-activated chloride channel in sarcoplasmic reticulum vesicles from rabbit skeletal muscle

1996 ◽  
Vol 270 (6) ◽  
pp. C1675-C1686 ◽  
Author(s):  
J. I. Kourie ◽  
D. R. Laver ◽  
G. P. Ahern ◽  
A. F. Dulhunty
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Lipid Bilayers ◽  
Chloride Channel ◽  
Single Channel ◽  
Single Channels ◽  
Channel Activity ◽  
Open Probability ◽  
Single Channel Activity ◽  
Maximum Conductance

A Ca(2+)-activated Cl- channel is described in sarcoplasmic reticulum (SR) enriched vesicles of skeletal muscle incorporated into lipid bilayers. Small chloride (SCl) channels (n = 20) were rapidly and reversibly activated when cis- (cytoplasmic) [Ca2+] was increased above 10(-7) M, with trans-(luminal) [Ca2+] at either 10(-3) or 10(-7) M. The open probability of single channels increased from zero when cis-[Ca2+] was 10(-7) M to 0.61 +/- 0.12 when [Ca2+] was 10(-4) M. High- and low-conductance levels in single-channel activity were activated at different cis-[Ca2+]. Channel openings to the maximum conductance, 65-75 pS (250/50 mM Cl-, cis/ trans), were active when cis-[Ca2+] was increased above 5 x 10(-6) M. In contrast to the maximum conductance, channel openings to submaximal levels between 5 and 40 pS were activated at a lower cis-[Ca2+] and dominated channel activity between 5 x 10(-7) and 5 x 10(-6) M. Activation of SCl channels was Ca2+ specific and not reproduced by cytoplasmic Mg2+ concentrations of 10(-3) M. We suggest that the SCl channel arises in the SR membrane. The Ca2+ dependence of this channel implies that it is active at [Ca2+] achieved during muscle contraction.

Abstract 407: Beta-blockers Reverse Beta Adrenergic Receptors Desensitization Under Hypoxia

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Yu Sun ◽  
Manveen K Gupta ◽  
Kate Stenson ◽  
Sathyamangla Prasad
Keyword(s):  
G Protein ◽  
Adrenergic Receptors ◽  
Plasma Membranes ◽  
Significant Loss ◽  
Beta Adrenergic Receptors ◽  
Beta Adrenergic ◽  
Β Blocker ◽  
Β Blockers ◽  
Underlying Mechanisms ◽  
Grk2 Expression

Hypoxia to heart or brain is a primary cause of heart failure or stroke. Studies have shown that hypoxia increases the beta-adrenergic receptors (βARs) phosphorylation and dysfunction (Cheong et. al., 2016). These observations provide evidence that βARs can directly be regulated by hypoxia but less is known about the underlying mechanisms. We postulated that hypoxia shifts the homeostasis between kinase and phosphatase driven mechanisms may underlie βAR dysfunction. β2AR HEK 293 cells were exposed to hypoxia (2% O 2 ) and assessed the mechanisms underlying desensitization (G-protein coupled receptor kinases, GRKs) and resensitization (Protein phosphatase 2A, PP2A). Six hours of hypoxia treatment resulted in increase of βAR phosphorylation and GRK2 expression, and interestingly, the internalization of phosphorylated β2AR is β-arrestin independent. Assessment of βAR phosphorylation in the plasma membrane and endosomal fractions surprisingly, showed marked increase in β2AR phosphorylation in the endosomal fraction. Furthermore, we also observed that receptor associated PP2A activity was inhibited in the endosomes following hypoxia with minimal changes of activity at the plasma membranes. At the same time, PI3Kγ activity markedly upregulated in the endosomes along with an increase of I2PP2A phosphorylation. Similarly subjecting normal mice to 20 hours of hypoxia resulted in significant cardiac dysfunction (% FS: Normoxia 38.83% vs. Hypoxia 32.38%, P=0.0055; % EF: Normoxia 69.71% vs. Hypoxia 60.76%,P=0.0105) and was associated with significant increase in β2AR phosphorylation associated with significant loss in βAR function as measured by G-protein coupling adenylyl cyclase activity. Given that β-blockers confer beneficial effects, we tested whether β-blocker (propranolol) would prevent βARs phosphorylation under hypoxia or normoxia. Consistently, β-blocker treatment in normoxia results in increased β2AR phosphorylation however, remarkably β-blocker treatment in hypoxia results in loss of β2AR phosphorylation, reduction in GRK2 expression and increase in βAR-associated PP2A. These studies show that agonist-independent hypoxia-driven β2AR dysfunction can be ameliorated by β-blockers and the underlying mechanisms for this unexpected findings will be discussed in the presentation. These findings have significant clinical implications as understanding these mechanisms could provide novel insights into the benefits provided by β-blockers.

Sign in / Sign up

Export Citation Format

Share Document