Recently a novel family of putative calcium-activated chloride channel (CLCA) proteins from bovine, murine and human origin have been identified by several laboratories (Gruber et al. 2000). We have previously reported the distribution of one of these homologues, the mCLCA3 protein, in peripheral tissues of the adult mouse (Shenton et al. 2002; Winpenny et al. 2002). However, very little is known about the role of this channel family in the mammalian brain. Here we provide the first evidence for the existence of the mCLCA3 channel in the adult mouse brain.
Our novel affinity-purified anti-mCLCA3 was generated in rabbits to a peptide sequence corresponding to the sequence KLETFKNAD (95-103) from the mCLCA3 amino acid sequence (GenBank Accession No. AB017156), following previously successful strategies (Chazot et al. 1994). All animals were humanely killed. The antibody was used to map the cellular distribution of the mCLCA3 channel protein in the adult murine brain using standard immunoblotting and immunohistochemical techniques (Thompson et al. 2002).
Initially, RT-PCR was performed using selective mCLCA3 oligonucleotide primers. The reaction identified a robust mRNA species for mCLCA3 in both adult mouse forebrain and cerebellum.
Immunoblotting experiments on mouse forebrain and cerebellar tissue homogenates detected major immunoreactive species present at 200 kDa, 120 kDa and 70 kDa. All these bands were blocked by prior incubation of the anti-mCLCA3 antibody with the immunising peptide. Immunohistochemical staining was carried out using Vectastain ABC kit according to manufacturer’s instructions together with anti-mCLCA3 as primary antibody. This technique demonstrated widespread neuronal labelling in many key structures within the brain, including the hippocampal formation and striatum and there was modest cortical and cerebellar staining. Immunoreactivity was negligible after preadsorption of the anti-mCLCA3 antibody with the immunising peptide. Highest expression was evident in the hippocampal formation with robust immunoreactive labelling of the cell bodies of the pyramidal neurons, granule cells and some hilar cells of the dentate gyrus.
Together these data demonstrate the expression of the mCLCA3 channel protein in neuronal tissue and suggest that the mCLCA3 channel may play a role in distinct populations of neurons and/or glia in the mammalian CNS.
This work was supported by the Wellcome Trust (059897) to J.P.W. & P.L.C.