Cochlea-Specific Deletion of Cav 1.3 Calcium Channels Arrests Inner Hair Cell Differentiation and Unravels Pitfalls of Conditional Mouse Models

Inner hair cell (IHC) Ca-v 1.3 Ca2+ channels are multifunctional channels mediating Ca2+ influx for exocytosis at ribbon synapses, the generation of Ca2+ action potentials in pre-hearing IHCs and gene expression. IHCs of deaf systemic Ca-v 1.3-deficient (Ca(v )1.3(-/-)) mice stay immature because they fail to up-regulate voltage- and Ca(2+)activated K+ (BK) channels but persistently express small conductance Ca2+-activated K+ (SK2) channels. In pre-hearing wildtype mice, cholinergic neurons from the superior olivary complex (SOC) exert efferent inhibition onto spontaneously active immature IHCs by activating their SK2 channels. Because Ca-v 1.3 plays an important role for survival, health and function of SOC neurons, SK2 channel persistence and lack of BK channels in systemic Ca(v )1.3(-/-)IHCs may result from malfunctioning neurons of the SOC. Here we analyze cochlea-specific Ca-v 1.3 knockout mice with green fluorescent protein (GFP) switch reporter function, Pax2::cre;Cacna1d-eGFP(flex/flex) and Pax2::cre;Cacna1d-eGFP(flex/-). Profound hearing loss, lack of BK channels and persistence of SK2 channels in Pax2::cre;Cacna1d-eGFP(flex/-) mice recapitulated the phenotype of systemic Ca(v )1.3(-/-) mice, indicating that in wildtype mice, regulation of SK2 and BK channel expression is independent of Ca-v 1.3 expression in SOC neurons. In addition, we noticed dose-dependent GFP toxicity leading to death of basal coil IHCs of Pax2::cre;Cacna1d-eGFP(flex/ flex) mice, likely because of high GFP concentration and small repair capacity. This and the slower time course of Pax2-driven Cre recombinase in switching two rather than one Cacna1d-eGFP (flex) allele lead us to study Pax2::cre;Cacna1d-eGFP(flex/ - )mice. Notably, control Cacna1d-eGFP(flex/-) IHCs showed a significant reduction in Ca-v 1.3 channel cluster sizes and currents, suggesting that the intronic construct interfered with gene translation or splicing. These pitfalls are likely to be a frequent problem of many genetically modified mice with complex or multiple gene-targeting constructs or fluorescent proteins. Great caution and appropriate controls are therefore required.