Calmodulin-dependent protein kinases mediate calcium-induced slow motility of mammalian outer hair cells

Abstract

Cochlear outer hair cells in vitro respond to elevation of intracellular calcium with slow shape changes over seconds to minutes (`slow motility'). This process is blocked by general calmodulin antagonists suggesting the participation of calcium/calmodulin-dependent enzymatic reactions. The present study proposes a mechanism for these reactions. Length changes of outer hair cells isolated from the guinea pig cochlea were induced by exposure to the calcium ionophore ionomycin. ATP levels remained unaffected by this treatment ruling out depletion of ATP (by activation of calcium-dependent ATPases) as a cause of the observed shape changes. Involvement of protein kinases was suggested by the inhibition of shape changes by K252a, a broad-spectrum inhibitor of protein kinase activity. Furthermore, the inhibitors ML-7 and ML-9 blocked the shape changes at concentrations compatible with inhibition of myosin light chain kinase (MLCK). KN-62, an inhibitor of Ca 2+/calmodulin-dependent protein kinase II (CaMKII), also attenuated the length changes. Inhibitors with selectivity for cyclic nucleotide-dependent protein kinases (H-89, staurosporine) were tested to assess potential additional contributions by such enzymes. The dose dependence of their action supported the notion that the most likely mechanism of slow motility involves phosphorylation reactions catalyzed by MLCK or CaMKII or both.