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 [G(Cl)(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-mu-M ionomycin. Bath application of the calmodulin (CaM) antagonists trifluoperazine, calmidazolium, or sphingosine (50-mu-M) reversibly inhibited G(Cl)(Ca), whereas the protein kinase C antagonists H7 and phloretin (50-mu-M) were without effect. This suggests that increases in intracellular Ca stimulate G(Cl)(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-mu-M) had no effect on G(Cl)(Ca). In contrast, stimulation of G(Cl)(Ca) by ionomycin was abolished when pipette solutions contained 10-mu-M CaMKII peptide inhibitor CaMKII-(273-302). The truncated peptide CaMKII-(284-302) (20-mu-M) lacks the CaMKII inhibitory domain and did not affect G(Cl)(Ca). These data suggest that CaM, acting through the multifunctional CaMKII, mediates the Ca-dependent stimulation of Cl conductance in colonic secretory cells.