Volume-regulated anion conductance in cultured rat cerebral astrocytes requires calmodulin activity.
We examined the calmodulin dependence of anion channel activation during hypo-osmotic swelling in rat cerebral astrocytes. Control cells bathed in iso-osmotic (290 mOsm) phosphate-buffered saline (PBS) and recorded using a patch electrode containing 140 mM KCl increased membrane conductance threefold over basal levels after 12 min in hypo-osmotic (200 mOsm) PBS. Cells injected with monoclonal anticalmodulin antibody demonstrated no increase in membrane conductance during a subsequent exposure to hypo-osmotic PBS. In contrast, cells iontophoretically injected with monoclonal antiglial fibrillary acidic protein antibody or with anticalmodulin antibody absorbed with an excess of free calmodulin demonstrated an increase in conductance during hypo-osmotic exposure similar to that of control cells. Conductance in iso-osmotic conditions was unchanged by antibody injection. Similar results were obtained when using patch electrode and bath solutions containing chloride as the only cell permeant ion, indicating a calmodulin-dependent anion current is activated with this degree of hypo-osmotic treatment. Western blots confirmed the specificity of the anticalmodulin and antiglial fibrillary acidic protein antibodies used in this study for proteins of 17 and 51 kD, respectively. In addition, in vitro studies demonstrated inhibition of the calmodulin-dependent activation of phosphodiesterase by the anticalmodulin antibody. Thus, binding of this antibody to calmodulin causes functional inhibition of calmodulin activity. No change in the intensity or cellular distribution of calmodulin immunostaining was observed during 30 min of hypo-osmotic exposure. However, increased immunostaining for activated calmodulin kinase IIalpha was observed after 10 min of hypo-osmotic exposure, suggesting initiation of calmodulin-dependent processes by cell swelling. The data indicate calmodulin activity is critical for activation of volume-regulated anion channels in rat cerebral astrocytes.
Olson
James E
JE
Department of Emergency Medicine, Wright State University School of Medicine, Cox Institute, Kettering, Ohio 45429, USA. james.olson@wright.edu
Li
Guang-Ze
GZ
Wang
Ling
L
Lu
Luo
L
D000818:Animals; D000831:Animals, Newborn; D027321:Anion Transport Proteins / Q000378:metabolism*; D000906:Antibodies / Q000494:pharmacology; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D048429:Cell Size / Q000502:physiology; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism / Q000494:pharmacology; D005904:Glial Fibrillary Acidic Protein / Q000037:antagonists & inhibitors / Q000378:metabolism; D007038:Hypotonic Solutions / Q000494:pharmacology; D007150:Immunohistochemistry; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009994:Osmolar Concentration; D010727:Phosphoric Diester Hydrolases / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology; D014882:Water-Electrolyte Balance / Q000502:physiology*
D027321:Anion Transport Proteins; D000906:Antibodies; D002147:Calmodulin; D002712:Chlorides; D005904:Glial Fibrillary Acidic Protein; D007038:Hypotonic Solutions; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C517675:Camk2a protein, rat; D010727:Phosphoric Diester Hydrolases