Modulation of K channels by coexpressed human alpha1C-adrenoceptor in Xenopus oocytes

1997 ◽  
Vol 272 (3) ◽  
pp. H1275-H1286
Author(s):  
G. N. Tseng ◽  
J. A. Yao ◽  
J. Tseng-Crank
Keyword(s):  
Protein Kinase ◽  
Model System ◽  
K Channel ◽  
K Channels ◽  
Channel Modulation ◽  
Channel Function ◽  
Adrenergic Modulation ◽  
Intracellular Ca ◽  
Dependent Protein ◽  
Multiple Receptor

alpha1-Adrenoceptors participate in the regulation of inotropy and chronotropy in the heart. Modulation of cardiac K-channel function plays an important role in these alpha1-adrenergic functions. Studies of the mechanisms of K-channel modulation by alpha1-adrenoceptors are hampered by the coexistence of multiple receptor and channel subtypes in the heart. We therefore used a model system of coexpressing a specific receptor (human alpha1c-adrenoceptor) and a K-channel clone (hIsK, rKv1.2, or rKv1.4) in oocytes. alpha1c-Adrenoceptor stimulation caused a rapid upregulation of hIsK by elevating the intracellular Ca concentration. At least part of this effect was due to an activation of calmodulin and Ca/calmodulin-dependent protein kinase II. On the other hand, alpha1c-adrenoceptor stimulation caused a slow downregulation of rKv1.2 and rKvl.4 by activating protein kinase C. The differential modulation of K channels by alpha1c-adrenoceptors demonstrated in our experiments corroborates the complexity of alpha1-adrenergic functions in the heart. Our results indicate that the oocyte model system can be a useful approach in studying alpha1-adrenergic modulation of ion-channel function and signal transduction.

Role of cAMP-dependent protein kinase in cAMP-mediated vasodilation

1992 ◽  
Vol 262 (2) ◽  
pp. H511-H516 ◽  
Author(s):  
J. Haynes ◽  
J. Robinson ◽  
L. Saunders ◽  
A. E. Taylor ◽  
S. J. Strada
Keyword(s):  
Protein Kinase ◽  
Pressor Response ◽  
Platelet Activating Factor ◽  
K Channel ◽  
Dependent Protein Kinase ◽  
K Channels ◽  
Pulmonary Vasodilation ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

In this study, the role of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) in cAMP-dependent relaxation was assessed in the isolated-perfused rat lung using a PKA inhibitor, Rp-cAMPS, 8-bromo-cAMP (8-BrcAMP), and the diterpene activator of adenylate cyclase (AC), forskolin (FSK). A role for K+ channels was also assessed with the nonselective K+ channel blocker, tetraethylammonium (TEA, 10 mM), and an ATP-sensitive K+ channel inhibitor, glibenclamide (GLI, 100 microM). Both 8-BrcAMP (0.1-1.0 mM) and RSK (0.1-10 microM) dose-dependently attenuated the peak pressor response to alveolar hypoxia (HPR). Rp-cAMPS potentiated the HPR and attenuated 8-BrcAMP-mediated vasodilation but had no effect on FSK-mediated vasodilation. FSK-mediated vasodilation was not mimicked by 1,9-dideoxy-FSK, which is biologically inactive on AC but alters K+ channels identically to FSK, nor was it attenuated by the platelet-activating factor antagonist SRI 63-441 or the cyclooxygenase inhibitor indomethacin. TEA, but not GLI, attenuated FSK-mediated vasodilation. Similarly, TEA attenuated 8-BrcAMP-mediated vasodilation. These results support roles for PKA and indirect gating of a non-ATP-sensitive K+ channel in mediating cAMP-dependent pulmonary vasodilation.

scholarly journals Estrogen Regulation of Genes Important for K+ Channel Signaling in the Arcuate Nucleus

Endocrinology ◽  
2007 ◽  
Vol 148 (10) ◽  
pp. 4937-4951 ◽  
Author(s):  
Troy A. Roepke ◽  
Anna Malyala ◽  
Martha A. Bosch ◽  
Martin J. Kelly ◽  
Oline K. Rønnekleiv
Keyword(s):  
Protein Kinase ◽  
Guinea Pig ◽  
Arcuate Nucleus ◽  
Neuronal Excitability ◽  
Estradiol Benzoate ◽  
K Channel ◽  
Pcr Analysis ◽  
Estrogen Regulation ◽  
K Channels ◽  
Channel Function

Estrogen affects the electrophysiological properties of a number of hypothalamic neurons by modulating K+ channels via rapid membrane actions and/or changes in gene expression. The interaction between these pathways (membrane vs. transcription) ultimately determines the effects of estrogen on hypothalamic functions. Using suppression subtractive hybridization, we produced a cDNA library of estrogen-regulated, brain-specific guinea pig genes, which included subunits from three prominent K+ channels (KCNQ5, Kir2.4, Kv4.1, and Kvβ1) and signaling molecules that impact channel function including phosphatidylinositol 3-kinase (PI3K), protein kinase Cε (PKCε), cAMP-dependent protein kinase (PKA), A-kinase anchor protein (AKAP), phospholipase C (PLC), and calmodulin. Based on these findings, we dissected the arcuate nucleus from ovariectomized guinea pigs treated with estradiol benzoate (EB) or vehicle and analyzed mRNA expression using quantitative real-time PCR. We found that EB significantly increased the expression of KCNQ5 and Kv4.1 and decreased expression of KCNQ3 and AKAP in the rostral arcuate. In the caudal arcuate, EB increased KCNQ5, Kir2.4, Kv4.1, calmodulin, PKCε, PLCβ4, and PI3Kp55γ expression and decreased Kvβ1. The effects of estrogen could be mediated by estrogen receptor-α, which we found to be highly expressed in the guinea pig arcuate nucleus and, in particular, proopiomelanocortin neurons. In addition, single-cell RT-PCR analysis revealed that about 50% of proopiomelanocortin and neuropeptide Y neurons expressed KCNQ5, about 40% expressed Kir2.4, and about 60% expressed Kv4.1. Therefore, it is evident that the diverse effects of estrogen on arcuate neurons are mediated in part by regulation of K+ channel expression, which has the potential to affect profoundly neuronal excitability and homeostatic functions, especially when coupled with the rapid effects of estrogen on K+ channel function.

scholarly journals ATP mediates both activation and inhibition of K(ATP) channel activity via cAMP-dependent protein kinase in insulin-secreting cell lines.

1989 ◽  
Vol 94 (4) ◽  
pp. 693-717 ◽  
Author(s):  
B Ribalet ◽  
S Ciani ◽  
G T Eddlestone
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Single Channel ◽  
K Channel ◽  
Channel Activity ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Insulin Secreting Cells ◽  
Dependent Protein ◽  
Inside Out

The single-channel recording technique was employed to investigate the mechanism conferring ATP sensitivity to a metabolite-sensitive K channel in insulin-secreting cells. ATP stimulated channel activity in the 0-10 microM range, but depressed it at higher concentrations. In inside-out patches, addition of the cAMP-dependent protein kinase inhibitor (PKI) reduced channel activity, suggesting that the stimulatory effect of ATP occurs via cAMP-dependent protein kinase-mediated phosphorylation. Raising ATP between 10 and 500 microM in the presence of exogenous PKI progressively reduced the channel activity; it is proposed that this inactivation results from a reduction in kinase activity owing to an ATP-dependent binding of PKI or a protein with similar inhibitory properties to the kinase. A model describing the effects of ATP was developed, incorporating these two separate roles for the nucleotide. Assuming that the efficacy of ATP in controlling the channel activity depends upon the relative concentrations of inhibitor and catalytic subunit associated with the membrane, our model predicts that the channel sensitivity to ATP will vary when the ratio of these two modulators is altered. Based upon this, it is shown that the apparent discrepancy existing between the sensitivity of the channel to low ATP concentrations in the excised patch and the elevated intracellular level of ATP may be explained by postulating a change in the inhibitor/kinase ratio from 1:1 to 3:2 owing to the loss of protein kinase after patch excision. At a low concentration of ATP (10-20 microM), a nonhydrolyzable ATP analogue, AMP-PNP, enhanced the channel activity when present below 10 microM, whereas the analogue blocked the channel activity at higher concentrations. It is postulated that AMP-PNP inhibits the formation of the kinase-inhibitor complex in the former case, and prevents phosphate transfer in the latter. A similar mechanism would explain the interaction between ATP and ADP which is characterized by enhanced activity at low ADP concentrations and blocking at higher concentrations.

Mechanism of activation by cGMP-dependent protein kinase of large Ca(2+)-activated K+ channels in mesangial cells

1996 ◽  
Vol 271 (5) ◽  
pp. C1669-C1677 ◽  
Author(s):  
J. D. Stockand ◽  
S. C. Sansom
Keyword(s):  
Protein Kinase ◽  
Mesangial Cells ◽  
Hill Coefficient ◽  
Voltage Sensitivity ◽  
Open Probability ◽  
Dependent Protein Kinase ◽  
K Channels ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Excised Patches

The patch clamp method was employed to establish the mechanism of regulation by guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) of large Ca(2+)-activated K+ channels (BKCa) in human mesangial cells. Dibutyryl cGMP (DBcGMP) significantly increased open probability (Po) of BKCa in the absence but not in the presence of staurosporine in cell-attached patches. In excised patches, BKCa was activated by simultaneous addition of MgATP plus cGMP but not cAMP plus MgATP. Activation by cGMP plus MgATP was blocked by KT-5823, an inhibitor of PKG, but not by KT-5720, an inhibitor of cAMP-dependent protein kinase (PKA). Thus a cGMP-specific endogenous kinase is associated with mesangial BKCa. In excised patches, exogenous PKG but not PKA or protein kinase C activated BKCa. The half-activation potential (V1/2), defined as the potential at which the Po = 0.5 with 1 microM Ca2+, was -34 and 42 mV for activated and inactivated BKCa, respectively; however, the gating charge (Zg), a measure of voltage sensitivity, was not affected by PKG. Similarly, the Ca1/2 (free Ca2+ concentration required to activate to Po = 0.5 at 40 mV) decreased from 1.74 to 0.1 microM on addition of PKG, but the Hill coefficient, a measure of Ca2+ sensitivity, was not affected. Activation of BKCa by PKG was heterogeneous with two populations: the majority (67%) activated by PKG and the minority unaffected. It is concluded that an endogenous PKG activates BKCa by decreasing the Ca2+ and voltage activation thresholds independently of sensitivities.

scholarly journals Potassium channel block by internal calcium and strontium.

1991 ◽  
Vol 97 (3) ◽  
pp. 627-638 ◽  
Author(s):  
C M Armstrong ◽  
Y Palti
Keyword(s):  
Time Course ◽  
External Solution ◽  
High Energy ◽  
K Channel ◽  
Giant Fiber ◽  
Distance Measurements ◽  
K Channels ◽  
High Energy Barrier ◽  
Intracellular Ca ◽  
A Site

We show that intracellular Ca blocks current flow through open K channels in squid giant fiber lobe neurons. The block has similarities to internal Sr block of K channels in squid axons, which we have reexamined. Both ions must cross a high energy barrier to enter the blocking site from the inside, and block occurs only with millimolar concentrations and with strong depolarization. With Sr (axon) or Ca (neuron) inside, IK is normal in time course for voltages less than about +50 mV; but for large steps, above +90 mV, there is a rapid time-dependent block or "inactivation." From roughly +70 to +90 mV (depending on concentration) the current has a complex time course that may be related to K accumulation near the membrane's outer surface. Block can be deepened by either increasing the concentration or the voltage. Electrical distance measurements suggest that the blocking ion moves to a site deep in the channel, possibly near the outer end. Block by internal Ca can be prevented by putting 10 mM Rb in the external solution. Recovery from block after a strong depolarization occurs quickly at +30 mV, with a time course that is about the same as that of normal K channel activation at this voltage. 20 mM Mg in neurons had no discernible blocking effect. The experiments raise questions regarding the relation of block to normal channel gating. It is speculated that when the channel is normally closed, the "blocking" site is occupied by a Ca ion that comes from the external medium.

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