CaMKII mediates stimulation of chloride conductance by calcium in T84 cells.
We used the secretory colonic cell line T84 to study the regulatory pathways controlling the Ca-stimulated Cl conductance [GCl(Ca)]. Under whole cell patch clamp, basal (unstimulated) current levels averaged 73 +/- 9 pA/20 pF (n = 93) and increased to 600 +/- 100 pA/20 pF (n = 53; at +100 mV) on exposure to 1-2 microM ionomycin. Bath application of the calmodulin (CaM) antagonists trifluoperazine, calmidazolium, or sphingosine (50 microM) reversibly inhibited GCl(Ca), whereas the protein kinase C antagonists H7 and phloretin (50 microM) were without effect. This suggests that increases in intracellular Ca stimulate GCl(Ca) via a CaM-dependent process rather than activating Cl channels directly. To assess the involvement of protein kinases in the Ca-dependent stimulation of Cl conductance, we employed pseudosubstrate peptide inhibitors of protein kinase C (PKC) and the Ca/CaM-dependent protein kinase II (CaMKII). Cellular concentrations of inhibitors during whole cell recording were estimated to be 4-20 times the inhibitory constant values for kinase inhibition observed in vitro. Pipette solutions containing the PKC peptide inhibitor PKC-(19-36) (7.5 microM) had no effect on GCl(Ca). In contrast, stimulation of GCl(Ca) by ionomycin was abolished when pipette solutions contained 10 microM CaMKII peptide inhibitor CaMKII-(273-302). The truncated peptide CaMKII-(284-302) (20 microM) lacks the CaMKII inhibitory domain and did not affect GCl(Ca). These data suggest that CaM, acting through the multifunctional CaMKII, mediates the Ca-dependent stimulation of Cl conductance in colonic secretory cells.
Worrell
R T
RT
Department of Physiology and Biophysics, University of Alabama, Birmingham 35294.
Frizzell
R A
RA
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002460:Cell Line; D018118:Chloride Channels; D003106:Colon; D007093:Imidazoles / Q000494:pharmacology; D007473:Ion Channels / Q000502:physiology*; D015759:Ionomycin / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008565:Membrane Proteins / Q000187:drug effects / Q000502:physiology*; D010446:Peptide Fragments / Q000494:pharmacology; D010693:Phloretin / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D013110:Sphingosine / Q000494:pharmacology; D014268:Trifluoperazine / Q000494:pharmacology
D002147:Calmodulin; D018118:Chloride Channels; D007093:Imidazoles; D007473:Ion Channels; D007546:Isoquinolines; D008565:Membrane Proteins; D010446:Peptide Fragments; D010879:Piperazines; D014268:Trifluoperazine; C031938:calmidazolium; D015759:Ionomycin; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011494:Protein Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D013110:Sphingosine; D010693:Phloretin; D002118:Calcium