Cochlea-Specific Deletion of Cav1.3 Calcium Channels Arrests Inner Hair Cell Differentiation and Unravels Pitfalls of Conditional Mouse Models

Abstract

Inner hair cell (IHC) Ca_v1.3 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_v1.3-deficient (Ca_v1.3^-/-) mice stay immature because they fail to up-regulate voltage- and Ca²⁺-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_v1.3 plays an important role for survival, health and function of SOC neurons, SK2 channel persistence and lack of BK channels in systemic Ca_v1.3^-/- IHCs may result from malfunctioning neurons of the SOC. Here we analyze cochlea-specific Ca_v1.3 knockout mice with green fluorescent protein (GFP) switch reporter function, <i>Pax2::cre;Cacna1d-eGFP</i> ^flex/flex <i>and Pax2::cre;Cacna1d-eGFP</i> ^flex/-. Profound hearing loss, lack of BK channels and persistence of SK2 channels in <i>Pax2::cre;Cacna1d-eGFP</i> ^flex/- mice recapitulated the phenotype of systemic Ca_v1.3^-/- mice, indicating that in wildtype mice, regulation of SK2 and BK channel expression is independent of Ca_v1.3 expression in SOC neurons. In addition, we noticed dose-dependent GFP toxicity leading to death of basal coil IHCs of <i>Pax2::cre;Cacna1d-eGFP</i> ^flex/flex mice, likely because of high GFP concentration and small repair capacity. This and the slower time course of <i>Pax2</i>-driven Cre recombinase in switching two rather than one <i>Cacna1d-eGFP^flex</i> allele lead us to study <i>Pax2::cre;Cacna1d-eGFP</i> ^flex/- mice. Notably, control <i>Cacna1d-eGFP^flex/-</i> IHCs showed a significant reduction in Ca_v1.3 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.