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Protein kinase inhibitor (PKI) is a potent endogenous inhibitor of the cyclic AMP (cAMP)-dependent protein kinase (PKA). It functions by binding the free catalytic (C) subunit with a high affinity and is also known to export nuclear C subunit to the cytoplasm. The significance of these actions with respect to PKI's physiological role is not well understood. To address this, we have generated by homologous recombination mutant mice that are deficient in PKIalpha, one of the three isoforms of PKI. The mice completely lack PKI activity in skeletal muscle and, surprisingly, show decreased basal and isoproterenol-induced gene expression in muscle. Further examination revealed reduced levels of the phosphorylated (active) form of the transcription factor CREB (cAMP response element binding protein) in the knockouts. This phenomenon stems, at least in part, from lower basal PKA activity levels in the mutants, arising from a compensatory increase in the level of the RIalpha subunit of PKA. The deficit in gene induction, however, is not easily explained by current models of PKI function and suggests that PKI may play an as yet undescribed role in PKA signaling.

In vertebrates, transduction of sound into an electrochemical signal is carried out by hair cells that rely on calcium to perform specialized functions. The apical surfaces of hair cells are surrounded by endolymphatic fluid containing calcium at concentrations that must be maintained by active transport. The mechanism of this transport is unknown, but an ATP-dependent pump is believed to participate. Mutation of the Atp2b2 gene that encodes plasma membrane calcium ATPase type 2 (PMCA2) produces the deaf, ataxic mouse: deafwaddler2J (dfw2J). We hypothesized that PMCA2 might transport calcium into the endolymph and that dfw2J mice would have low endolymph calcium concentrations, possibly contributing to their deafness and ataxia. First, using immunocytochemistry, we demonstrated that PMCA2 is present in control mice inner and outer hair cell stereocilia where it could pump calcium into the endolymph and that PMCA2 is absent in dfw2J stereocilia. Second, using an aspirating microelectrode and calcium-sensitive fluorescent dye, we found that dfw2J mice endolymph calcium concentrations are significantly lower than those of control mice. These findings suggest that PMCA2, located in hair cell stereocilia, contributes significantly to endolymph calcium maintenance.