Mechanism of the Inhibition of Ca2+-Activated Cl− Currents by Phosphorylation in Pulmonary Arterial Smooth Muscle Cells
| Autor: | Jeff E. Angermann, Normand Leblanc, James L. Kenyon, Amy R. Sanguinetti, Iain A. Greenwood |
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| Rok vydání: | 2006 |
| Předmět: |
Patch-Clamp Techniques
Physiology Adenylyl Imidodiphosphate Myocytes Smooth Muscle chemistry.chemical_element Gating Pulmonary Artery Calcium Models Biological Article Membrane Potentials 03 medical and health sciences chemistry.chemical_compound Adenosine Triphosphate 0302 clinical medicine Chlorides Chloride Channels Animals Computer Simulation Patch clamp Phosphorylation 030304 developmental biology Membrane potential 0303 health sciences Chemistry Pipette Articles Kinetics Biochemistry Biophysics Chloride channel Rabbits Ion Channel Gating Adenosine triphosphate 030217 neurology & neurosurgery |
| Zdroj: | The Journal of General Physiology |
| ISSN: | 1540-7748 0022-1295 |
| DOI: | 10.1085/jgp.200609507 |
| Popis: | The aim of the present study was to provide a mechanistic insight into how phosphatase activity influences calcium-activated chloride channels in rabbit pulmonary artery myocytes. Calcium-dependent Cl- currents (I(ClCa)) were evoked by pipette solutions containing concentrations between 20 and 1000 nM Ca2+ and the calcium and voltage dependence was determined. Under control conditions with pipette solutions containing ATP and 500 nM Ca2+, I(ClCa) was evoked immediately upon membrane rupture but then exhibited marked rundown to approximately 20% of initial values. In contrast, when phosphorylation was prohibited by using pipette solutions containing adenosine 5'-(beta,gamma-imido)-triphosphate (AMP-PNP) or with ATP omitted, the rundown was severely impaired, and after 20 min dialysis, I(ClCa) was approximately 100% of initial levels. I(ClCa) recorded with AMP-PNP-containing pipette solutions were significantly larger than control currents and had faster kinetics at positive potentials and slower deactivation kinetics at negative potentials. The marked increase in I(ClCa) was due to a negative shift in the voltage dependence of activation and not due to an increase in the apparent binding affinity for Ca2+. Mathematical simulations were carried out based on gating schemes involving voltage-independent binding of three Ca2+, each binding step resulting in channel opening at fixed calcium but progressively greater "on" rates, and voltage-dependent closing steps ("off" rates). Our model reproduced well the Ca2+ and voltage dependence of I(ClCa) as well as its kinetic properties. The impact of global phosphorylation could be well mimicked by alterations in the magnitude, voltage dependence, and state of the gating variable of the channel closure rates. These data reveal that the phosphorylation status of the Ca2+-activated Cl- channel complex influences current generation dramatically through one or more critical voltage-dependent steps. |
| Databáze: | OpenAIRE |
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