Dual regulation of the ATP-sensitive potassium channel by caffeine

2007 ◽  
Vol 292 (6) ◽  
pp. C2239-C2258 ◽  
Author(s):  
Xia Mao ◽  
Yongping Chai ◽  
Yu-Fung Lin
Keyword(s):  
Single Channel ◽  
Regulatory Protein ◽  
Protein S ◽  
Dependent Manner ◽  
Sulfonylurea Receptor ◽  
Concentration Dependent Manner ◽  
Stimulatory Action ◽  
Hek 293 ◽  
Single Channel Currents ◽  
Channel Currents

ATP-sensitive potassium (KATP) channels couple cellular metabolic status to changes in membrane electrical properties. Caffeine (1,2,7-trimethylxanthine) has been shown to inhibit several ion channels; however, how caffeine regulates KATP channels was not well understood. By performing single-channel recordings in the cell-attached configuration, we found that bath application of caffeine significantly enhanced the currents of Kir6.2/SUR1 channels, a neuronal/pancreatic KATP channel isoform, expressed in transfected human embryonic kidney (HEK)293 cells in a concentration-dependent manner. Application of nonselective and selective phosphodiesterase (PDE) inhibitors led to significant enhancement of Kir6.2/SUR1 channel currents. Moreover, the stimulatory action of caffeine was significantly attenuated by KT5823, a specific PKG inhibitor, and, to a weaker extent, by BAPTA/AM, a membrane-permeable Ca2+ chelator, but not by H-89, a selective PKA inhibitor. Furthermore, the stimulatory effect was completely abrogated when KT5823 and BAPTA/AM were co-applied with caffeine. In contrast, the activity of Kir6.2/SUR1 channels was decreased rather than increased by caffeine in cell-free inside-out patches, while tetrameric Kir6.2LRKR368/369/370/371AAAA channels were suppressed regardless of patch configurations. Caffeine also enhanced the single-channel currents of recombinant Kir6.2/SUR2B channels, a nonvascular smooth muscle KATP channel isoform, although the increase was smaller. Moreover, bidirectional effects of caffeine were reproduced on the KATP channel present in the Cambridge rat insulinoma G1 (CRI-G1) cell line. Taken together, our data suggest that caffeine exerts dual regulation on the function of KATP channels: an inhibitory regulation that acts directly on Kir6.2 or some closely associated regulatory protein(s), and a sulfonylurea receptor (SUR)-dependent stimulatory regulation that requires cGMP-PKG and intracellular Ca2+-dependent signaling.

Ammonium ion enhances the calcium-dependent gating of a mammalian large conductance, calcium-sensitive K+ channel

2001 ◽  
Vol 79 (11) ◽  
pp. 919-923 ◽  
Author(s):  
Andrew P Braun
Keyword(s):  
Single Channel ◽  
Ammonium Ion ◽  
K Channel ◽  
Free Calcium ◽  
Dependent Manner ◽  
Open Probability ◽  
Concentration Dependent Manner ◽  
Hek 293 ◽  
Calcium Dependent ◽  
Single Channel Currents

We observed that the current amplitude and activation of expressed, mouse brain large conductance, calcium-sensitive K+ channels (BKCa channels) may be reversibly enhanced following addition of low concentrations of the weakly permeant cation NH4+ to the cytoplasmic face of the channel in excised, inside-out membrane patches from HEK 293 cells. Conductance-voltage relations were left-shifted along the voltage axis by addition of NH4Cl in a concentration-dependent manner, with an EC50 of 18.5 mM. Furthermore, this effect was observed in the presence of cytosolic free calcium (~1 µM), but was absent in a cytosolic bath solution containing nominally zero free calcium (e.g., 5 mM EGTA only), a condition under which these channels undergo largely voltage-dependent gating. Recordings of single BKCa channel events indicated that NH4+ increased the channel open probability of single channel activity ~3-fold, but did not alter the amplitude of single channel currents. These findings suggest that the calcium-sensitive gating of mammalian BKCa channels may be modified by other ions present in cytosolic solution.Key words: potassium channel, calcium, modulation, electrophysiology.

scholarly journals The nature and origin of spontaneous noise in G protein-gated ion channels.

1991 ◽  
Vol 97 (6) ◽  
pp. 1279-1293 ◽  
Author(s):  
K Okabe ◽  
A Yatani ◽  
A M Brown
Keyword(s):  
Ion Channels ◽  
G Protein ◽  
Single Channel ◽  
Coupled Systems ◽  
Dependent Manner ◽  
Protein Receptor ◽  
Spontaneous Noise ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Inside Out

Arrival of agonist is generally thought to initiate the signal transduction process in G protein-receptor coupled systems. However, the muscarinic atrial K+ (K+[ACh]) channel opens spontaneously in the absence of applied agonist, giving a noisy appearance to the current records. We investigated the nature and origin of the noise by measuring single channel currents in cell-attached or excised, inside-out membrane patches. Guanosine triphosphate (GTP) produced identical single channel currents in a concentration- and Mg(2+)-dependent manner in the presence or absence of carbachol, but the requirements for GTP were greater in the absence of agonist. Hence the agonist-independent currents appeared to be produced by an endogenous G protein, Gk. This prediction was confirmed when an affinity-purified, sequence-specific Gi-3 alpha antibody or pertussis toxin (PTX) blocked the agonist-independent currents. Candidate endogenous agonists were ruled out by the lack of effect of their corresponding antagonists. Thus agonist-independent currents had the same nature as agonist-dependent K+[ACh] currents and seemed to originate in the same way. We have developed a hypothesis in which agonist-free, empty receptors prime Gk with GTP and Gk activates atrial K+ [ACh] channels producing basal currents or noise. Agonist-independent activation by G proteins of effectors including ion channels appears to be a common occurrence.

Effect of a novel BKCa opener on BKCa currents and contractility of the rabbit corpus cavernosum

2016 ◽  
Vol 310 (4) ◽  
pp. C284-C292 ◽  
Author(s):  
K. I. Hannigan ◽  
R. J. Large ◽  
E. Bradley ◽  
M. A. Hollywood ◽  
G. P. Sergeant ◽  
...  
Keyword(s):  
Single Channel ◽  
Corpus Cavernosum ◽  
Isometric Tension ◽  
Mechanical Activity ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Rabbit Corpus Cavernosum ◽  
Current Clamp Mode ◽  
Single Channel Currents ◽  
Spontaneous Contractions

Large-conductance Ca2+-activated K+ (BKCa) channels are thought to play a key role in the regulation of corpus cavernosum smooth muscle (CCSM) excitability. Few BKCa channel openers have been accepted for clinical development. The effect of the novel BKCa channel opener GoSlo-SR5-130 on electrical activity in isolated rabbit CCSM cells and mechanical activity in strips of rabbit CCSM was examined. Single-channel currents were observed in inside-out patches. These channels were sensitive to Ca2+, blocked by penitrem A, and had a conductance of 291 ± 20 pS ( n = 7). In the presence of GoSlo-SR5-130, the number of open BKCa channels increased. Using voltage-ramp protocols, GoSlo-SR5-130 caused currents to activate at more negative potentials in a concentration-dependent manner, shifting the half-maximal activation voltage potential to the left on the voltage axis. Therefore, BKCa channels were open within the physiological range of membrane potentials in the presence of GoSlo-SR5-130. GoSlo-SR5-130 also resulted in an increase in the activity of spontaneous transient outward currents in myocytes isolated from CCSM, and this effect was reversed by iberiotoxin. In current-clamp mode, GoSlo-SR5-130 hyperpolarized the cell membrane. Isometric tension recording of strips of rabbit corpus cavernosum showed that GoSlo-SR5-130 inhibited spontaneous contractions in a concentration-dependent manner. This effect was reversed in the presence of iberiotoxin, suggesting that GoSlo-SR5-130 exerts its effect through BKCa channels. These findings suggest that GoSlo-SR5-130 is an effective tool for the study of BKCa channels and that these channels can modulate CCSM activity and are possible targets for the treatment of erectile dysfunction.

Proton Modulation of α1β3δ GABAA Receptor Channel Gating and Desensitization

2004 ◽  
Vol 92 (3) ◽  
pp. 1577-1585 ◽  
Author(s):  
Hua-Jun Feng ◽  
Robert L. Macdonald
Keyword(s):  
Steady State ◽  
Single Channel ◽  
Dependent Manner ◽  
Human Embryonic Kidney Cells ◽  
Receptor Isoforms ◽  
Long Duration ◽  
A Subunit ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Receptor Channel

αβγ GABAA receptor currents are phasic and desensitizing, whereas αβδ GABAA receptor currents are tonic and have no fast desensitization. αβγ receptors are subsynaptic and mediate phasic inhibition, whereas αβδ receptors are extra- or perisynaptic and mediate tonic inhibition. Given the different roles of these GABAA receptor isoforms and the fact that GABAA receptors are allosterically regulated by extracellular pH in a subunit-dependent manner, we compared the effects of changing pH on rat δ or γ2L subunit–containing GABAA receptor currents. Human embryonic kidney cells (HEK293T) were transfected with cDNAs encoding rat α1, β3, γ2L, or δ GABAA receptor subunits in several binary and ternary combinations, and whole cell and single channel patch-clamp recordings were obtained. Lowering pH substantially enhanced α1β3 receptor currents. This effect was significantly more pronounced for ternary α1β3δ receptors, whereas ternary α1β3γ2L receptors were relatively insensitive to lowered pH. Lowering pH did not affect the extent of desensitization of α1β3 and α1β3γ2L receptor currents, but significantly increased the extent of desensitization of α1β3δ receptor currents. Lowering pH prolonged deactivation of α1β3 and α1β3δ receptor currents and enhanced the “steady-state” currents of α1β3δ receptors evoked by long-duration (28 s) GABA applications. Lowering pH significantly increased mean open duration of α1β3δ steady-state single channel currents due to introduction of a longer-duration open state, suggesting that low pH enhances α1β3δ receptor steady-state currents by modifying GABAA receptor gating properties.

scholarly journals Ion conductance and selectivity of single calcium-activated potassium channels in cultured rat muscle.

1984 ◽  
Vol 84 (1) ◽  
pp. 1-23 ◽  
Author(s):  
A L Blatz ◽  
K L Magleby
Keyword(s):  
Dissociation Constant ◽  
Single Channel ◽  
Reversal Potential ◽  
Ion Concentration ◽  
Dependent Manner ◽  
Apparent Dissociation Constant ◽  
Rat Muscle ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Channel Currents

The conductance and selectivity of the Ca-activated K channel in cultured rat muscle was studied. Shifts in the reversal potential of single channel currents when various cations were substituted for Ki+ were used with the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The selectivity was Tl+ greater than K+ greater than Rb+ greater than NH4+, with permeability ratios of 1.2, 1.0, 0.67, and 0.11. Na+, Li+, and Cs+ were not measurably permeant, with permeabilities less than 0.05 that of K+. Currents with the various ions were typically less than expected on the basis of the permeability ratios, which suggests that the movement of an ion through the channel was not independent of the other ions present. For a fixed activity of Ko+ (77 mM), plots of single channel conductance vs. activity of Ki+ were described by a two-barrier model with a single saturable site. This observation, plus the finding that the permeability ratios of Rb+ and NH+4 to K+ did not change with ion concentration, is consistent with a channel that can contain a maximum of one ion at any time. The empirically determined dissociation constant for the single saturable site was 100 mM, and the maximum calculated conductance for symmetrical solutions of K+ was 640 pS. TEAi+ (tetraethylammonium ion) reduced single channel current amplitude in a voltage-dependent manner. This effect was accounted for by assuming voltage-dependent block by TEA+ (apparent dissociation constant of 60 mM at 0 mV) at a site located 26% of the distance across the membrane potential, starting at the inner side. TEAo+ was much more effective in reducing single channel currents, with an apparent dissociation constant of approximately 0.3 mM.

scholarly journals Ca2+ signalling in K562 human erythroleukaemia cells: effect of dimethyl sulphoxide and role of G-proteins in thrombin- and thromboxane A2-activated pathways

1995 ◽  
Vol 312 (1) ◽  
pp. 151-158 ◽  
Author(s):  
C P Thomas ◽  
M J Dunn ◽  
R Mattera
Keyword(s):  
G Proteins ◽  
Pcr Amplification ◽  
Thromboxane A2 ◽  
K562 Cells ◽  
Protein S ◽  
Term Treatment ◽  
Dependent Manner ◽  
Short Term Treatment ◽  
Concentration Dependent Manner ◽  
Prostaglandin F2 Alpha

The human leukaemic cell line K562 is a pluripotent stem cell with the potential to mature along a megakaryocytic or erythroid line. In these cells, thrombin and U46619 (9,11-dideoxy-9 alpha, 11 alpha-methanoepoxy prostaglandin F2 alpha), a thromboxane A2 analogue, increased intracellular Ca2+ in a rapid and concentration-dependent manner. The peak transient observed with both thrombin and U46619 was preserved upon stimulation in the absence of extracellular calcium and blunted with phorbol myristate acetate, suggestive of activation of phospholipase C. Short-term treatment with leupeptin abolished the calcium response to thrombin, but did not alter that to U46619. Both pertussis toxin (PT) and DMSO pretreatment inhibited thrombin- but not U46619-stimulated intracellular calcium elevation, indicating that these agonists signal through different G-proteins. Western blot analysis of crude membranes from K562 cells revealed the presence of G12 alpha and G13 alpha; the other known PT-substrates, Gi1 alpha and G0 alpha, were not detected. Consistent with this observation, ADP-ribosylation experiments revealed the presence of two PT substrates which co-migrated with human erythrocyte G12 alpha and G13 alpha. An antibody raised against Gq/11 alpha, a subfamily of G-protein alpha subunits unmodified by PT, specifically recognized 42 kDa protein(s) in K562 cells. PCR amplification of reverse-transcribed K562 RNA followed by DNA sequencing showed that these cells express messages for both Gq alpha and G11 alpha. Treatment of K562 cells with DMSO reduced the levels of thrombin receptor mRNA, without simultaneous changes in the expression of G12 alpha and G13 alpha. We have thus identified Ca(2+)-mobilizing agonists and related G-proteins in K562 cells, together with changes induced by DMSO in this signalling pathway.

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