Abstract/Summary

CACHD1 is classified as a member of the α2δ protein family due to their shared organisation of structural motifs and domains. However, unlike α2δ, CACHD1 has been characterised as a modulator of the CaV3 low-voltage-activated voltage-gated Ca2+ channels (LVA VGCCs), distinguishing it as the sole auxiliary subunit of LVA VGCCs. As a relatively unstudied protein, CACHD1 research has primarily focused on its functional effects on LVA VGCCs and its potential role in disease. This study represents the first investigation into the mechanisms by which specific motifs govern CACHD1 modulation of LVA VGCCs, and its trafficking pathways. This thesis tested the hypothesis that structural motifs within CACHD1 play crucial roles in regulating its expression, sub-cellular localisation, trafficking, and function as a modulator of LVA VGCCs. This study first characterised the role of the variant MIDAS motif (D234xGxS) on CACHD1 expression, sub-cellular localisation, and function as a modulator of CaV3 channels, using western blotting, immunocytochemistry, and whole-cell patch clamp electrophysiology. Interestingly, mutagenesis of the G236 residue to Ser showed similar CACHD1 expression and sub-cellular localisation in the absence of CaV3.1 compared to wild-type CACHD1 (CACHD1- wt). Moreover, like CACHD1-wt, CACHD1-G236S significantly increased the current density and Gmax of rat CaV3.1 (rCaV3.1) VGCCs. In contrast, mutating all three key residues to Ala (DxGxS to AxAxA) led to significant reduction in CACHD1 expression levels and disrupted trafficking to cell surface. Additionally, the AxAxA mutant resulted in a significant decrease in rCaV3.1 currents and Gmax, underscoring the important role of an intact MIDAS motif in CACHD1 function. Next, this study investigated the internalisation, and post-endocytic mechanisms used by CACHD1. The use of endocytic inhibitors showed that methyl-β-cyclodextrin (MβCD), a cholesterol depleting agent, inhibited CACHD1 internalisation. Moreover, cholesterol recovery post-MβCD treatment restored CACHD1 internalisation, suggesting a caveolae pathway dependent on cholesterol was used by CACHD1. Additionally, CACHD1 co-localised with EEA1, Rab5, and Rab11, indicating CACHD1 recycling back to the membrane via a Rab11- associated pathway. Further analysis identified a tyrosine internalisation motif (Y1197STM) present in the intracellular C-terminal tail of CACHD1. Mutagenesis of the Y1197 residue to Ala and Phe showed that the Y1197F slowed CACHD1 internalisation. Surprisingly, the removal of the C-terminal tail, thus the YSTM signal motif, had no discernible effect on CACHD1 internalisation, indicating the involvement of multiple motifs. Moreover, removal of the transmembrane domain led to intracellular retention of CACHD1, but modification of the natural signal peptide to Igκ signal peptide facilitated protein secretion, enabling the purification of soluble CACHD1 protein.