In Silico Binding Mode Analysis of Blarina Paralytic Peptides with the Human T-Type Ca Channel hCav3.2

<i>Blarina</i> paralytic peptides (BPPs), neurotoxins from shrew saliva that paralyze mealworms, share high sequence similarity with human synenkephalin [1-53] (hSYN), a peptide released from proenkephalin together with opioid peptides that mediate analgesic and antidepressant effects in the brain. Both synthetic BPP2 and hSYN induce a hyperpolarizing shift in the human T-type voltage-gated calcium channel (hCa_v3.2) at sub-micromolar concentrations, although only BPP2 causes paralysis in insects. To gain insight into the functions of these insectivorous animal-specific neurotoxins and the largely uncharacterized brain peptides, we investigated the structure prediction of BPPs and SYNs and their interactions with hCa_v3.2. AlphaFold 3 modeling complemented available cryo-EM data and accurately reproduced the overall channel architecture; however, this inactivated-state model proved unsuitable for predicting agonistic binding of BPPs and SYNs. In contrast, docking simulations using an activated-state hCa_v3.2 homology model revealed distinct ligand-dependent differences in binding energies, affinity, and conformational flexibility. Notably, the C-terminal tail of BPPs-particularly its variable length and flexibility-was identified as a key determinant for the interactions with the S4 voltage-sensing domain of the channel. These findings provide new insights into the evolutionary adaptation of venom peptides in mammals and into potential therapeutic strategies targeting neurological disorders.