<i>Pasteurella multocida</i> toxin disrupts the respiratory epithelial barrier by utilizing the T-type voltage-gated Ca²⁺ channel Ca_V3.1 as a candidate receptorPasteurella multocida toxin disrupts the respiratory epithelial barr

<i>Pasteurella multocida</i> toxin (PMT) is an exotoxin produced by several members of the zoonotic respiratory pathogen <i>P. multocida</i>. The role of PMT in disrupting the mammalian respiratory barrier remains to be elucidated. In this study, we discovered that inoculation of recombinantly expressed PMT increased the permeability of the respiratory epithelial barrier in mouse and respiratory cell models. This was evidenced by a decreased expression of tight junctions (ZO-1, occludin) and adherens junctions (β-catenin, E-cadherin), as well as enhanced cytoskeletal rearrangement. In mechanism, we demonstrated that PMT inoculation induced cytoplasmic Ca²⁺ inflow, leading to an imbalance of cellular Ca²⁺ homeostasis and endoplasmic reticulum stress. This process further stimulated the RhoA/ROCK signaling, promoting cytoskeletal rearrangement and reducing the expression of tight junctions and adherens junctions. Notably, the T-type voltage-gated Ca²⁺ channel Ca_V3.1 was found to participate in PMT-induced cytoplasmic Ca²⁺ inflow. Knocking out Ca_V3.1 significantly reduced the cytotoxicity induced by PMT on swine respiratory epithelial cells and mitigated cytoplasmic Ca²⁺ inflow stimulated by PMT. Further analysis identified Ser (aa92), Glu (aa155), Tyr (aa167), and Leu (aa448) as crucial sites utilized by PMT to interact with Ca_V3.1. These findings suggest Ca_V3.1 serves as an important host receptor of PMT and contributes to PMT-induced respiratory epithelial barrier disruption.