CACHD1

T-channels are best known for the generation of low-threshold spikes (LTS), responsible for neuronal burst firing, however, recent studies support a crucial role for T-channels in tonic, single spiking phases, reinterpreting the importance of T-channels in controlling neuronal excitability at membrane potentials where T-channels were originally thought to be fully inactivated. T-channels have been shown to play a critical role in facilitating burst firing of neurons in TLE. Cache domain containing 1 protein (CACHD1) is a protein that has predicted structural similarities to members of the α2δ voltage-gated calcium channel auxiliary subunit family. CACHD1 protein is highly expressed in the CNS, with high expression in both rat and human hippocampus. We recently showed that CACHD1 over expression increases T- current amplitudes and increases cultured hippocampal neuron, suggesting that it is a modulator of T-type calcium channels. Understanding the importance of CACHD1 in modulating T-channel activity and determining if CACHD1 plays a role in increasing neuronal hyperexcitability associated with epilepsy is highly novel and significant. Using CRISPR we have generated a CACHD1 knockout mouse and will continue to explore the role of CACHD1 in modulating T-channel activity in epilepsy.

We will begin to define the mechanism by which CACHD1 regulates T-channel expression and activity. We will determine if the MIDAS motif in the extracellular domain of CACHD1 is important for Ca_v3.1 modulation, if a tyrosine-based endocytic signal within the intracellular domain of CACHD1 is important for its internalization and if an aspartic acid residue on domain I of Ca_v3.1 is the CACHD1 interaction site.

Since T-channels are associated with absence epilepsy we plan to determine if knockout of CACHD1 (CACHD1 KO) reduces T-channel activity and excitability of thalamocortical relay neurons (TC) and reticular neurons (nRT). We also plan to determine if a knockout of CACHD1 in the stargazer (stg) mouse model of absence epilepsy reduces T-channel currents, suppresses burst firing of TC and nRT neurons and reduces and/or prevents onset of absence seizures.

T-channels also play a major role in controlling the excitability of neurons, and have increased expression and activity in temporal lobe epilepsy. We plan to investigate knockdown of CACHD1 decreases T- currents, reduces subiculum neuron excitability and delays, or event prevents, seizure onset and suppresses seizure severity and frequency in a mouse model of temporal lobe epilepsy.