Localization and expression of CaBP1/caldendrin in the mouse brain.

In the nervous system, the generation and propagation of Ca2+ signals is crucial for the control of neuronal excitability, gene expression, and synaptic plasticity(Augustine et al., 2003). The ubiquitous Ca2+ sensing protein, calmodulin (CaM) is thought to be a major player in this process. CaM is the prototypical member of the superfamily of EF-hand Ca2+ binding proteins, so named because they contain one or more helix-loop-helix domains that coordinate a Ca2+ ion (Kretsinger, 1976). CaM modulates the activity of numerous effector proteins (Chin and Means, 2000) and is an essential regulator of neuronal development (Bolsover, 2005) and synaptic plasticity (Xia and Storm, 2005). A subset of EF-hand proteins related to CaM is expressed mainly in neurons (Burgoyne et al., 2004). Most similar to CaM are the CaBPs (CaBP1-8), which are found primarily in the brain, retina, and inner ear (Haeseleer et al., 2000, Laube et al., 2002, Yang et al., 2006, Cui et al., 2007). A wealth of evidence supports a role for CaBPs as regulators of voltage-gated Cav Ca2+ channels. CaBP4 potentiates the activity of Cav1.4 (Haeseleer et al., 2004, Shaltiel et al., 2012) and Cav1.3 Ca2+ channels (Yang et al., 2006, Cui et al., 2007, Lee et al., 2007), and CaBP2 enhances the function of Cav1.3 channels (Schrauwen et al., 2012). Cav1.4 and Cav1.3 are the primary Cav channels that regulate neurotransmitter release from retinal photoreceptors and cochlear inner hair cells, respectively (Platzer et al., 2000, Mansergh et al., 2005). Human mutations in the genes encoding CaBP4 and CaBP2 impair Cav1 regulation and cause vision and hearing impairment, respectively (Zeitz et al., 2006, Littink et al., 2009, Schrauwen et al., 2012, Shaltiel et al., 2012). Unlike CaBP4 and CaBP2, CaBP1 is highly expressed in the brain (Haeseleer et al., 2000). Alternative splicing of the N-terminal domain gives rise to multiple variants (CaBP1-S, CaBP1-L, and caldendrin) (Seidenbecher et al., 1998, Haeseleer et al., 2000). CaBP1 has powerful effects in opposing CaM regulation of Cav channels(Lee et al., 2002). For the major Cav1 channels in the brain (Cav1.2 and Cav1.3), CaBP1 competes with CaM for binding to the channel and facilitates channel opening by preventing Ca2+/CaM-dependent inactivation (Lee et al., 2002, Zhou et al., 2004, Zhou et al., 2005, Yang et al., 2006, Cui et al., 2007, Findeisen and Minor, 2010, Oz et al., 2011). CaBP1 also binds to and regulates inositol 1,4,5 – trisphosphate receptors (IP3Rs) (Yang et al., 2002, Haynes et al., 2004). The largest CaBP1 variant, caldendrin, was initially discovered as a component of the postsynaptic membrane of excitatory synapses in the brain (Seidenbecher et al., 1998), where it may interact with the cytoskeleton (Seidenbecher et al., 2004), A-kinase anchoring protein (AKAP)79/150 (Gorny et al., 2012), Cav1.2 (Tippens and Lee, 2007), and/or Jacob, a protein that couples N-methyl-D-aspartate (NMDA) receptor signaling to the nucleus (Dieterich et al., 2008). The above findings suggest a role for CaBP1 and caldendrin in regulating neuronal Ca2+ signaling, but the neurophysiological significance of these proteins in vivo is unknown. To fill this gap in knowledge, we generated mice with targeted inactivation of the CaBP1/caldendrin gene (C-KO). While previous studies have characterized the distribution of CaBP1 and caldendrin in the nervous system of rat (Laube et al., 2002) and human (Bernstein et al., 2003), similar analyses in the mouse are necessary to understand phenotypes of the C-KO mice. Therefore, we used molecular, biochemical, and immunocytochemical strategies to characterize the expression and localization of CaBP1 and caldendrin in the mouse brain.