Structure‐Function Relationship for Spermine‐mediated Inward Rectification of Transient Receptor Potential Canonical (TRPC) 4 Channels.
Authors: Park Christine Haewon , Kim Jinsung , So Insuk 
Published: April 2020

Transient Receptor Potential Canonical (TRPC) channels constitute calcium‐permeable, non‐selective cation channels in mammalian cells. The expression of TRPC4 channels can be found in gastrointestinal (GI) tract, endometrium, ventricular myocytes, vascular smooth muscle cells, endothelium and many others. In such tissues, it is well‐known that TRPC4 channels exert membrane excitatory effect due to inwardly‐rectifying nature of current‐voltage (I‐V) relationship of the channels. Based on the electrophysiological similarity between inwardly‐rectifying potassium channel and TRPC4 channel, we investigated the molecular mechanism of inward‐rectification of TRPC4 channels, especially by intracellular magnesium and polyamine, which is two major cytoplasmic blocking agent of IRKs.Based on the pre‐established theory for inward‐rectification of IRKs, we have constructed a number of mutant TRPC4 channels whose acidic amino acid (glutamate or aspartate) at cytoplasmic side (C‐terminus) was changed to alanine. Such mutant channels were overexpressed in HEK293 cells and the activity and efficacy of cytoplasmic blocking action of spermine was measured using whole‐cell patch‐clamp technique. Structural analysis was based on published Cryo‐EM structure of mouse TRPC4 channels (PDB Accession Number: 5z96). The modeled 3D structures of TRPC4 mutants were visualized and analyzed using the PyMOL software. For the calculation of electrical distance, two‐barrier model with one reaction‐intermediate potential was conveyed.As a result, the electrical distance of spermine demonstrated that spermine barely penetrates into the pore but rather exerts its action on the entrance of putative inner vestibule of the channel. The voltage‐dependency of Kd value was not significant compared to other well‐known blocking agents such as proton in Na channels and polyamine in IRKs. Among various mutant channels, D629A, E648A, E728A and E729A channels showed significant recovery from spermine block, demonstrating that such residues are crucial for spermine‐mediated inward rectification. Identification of such residues in Cryo‐EM structure of TRPC4 channel showed rather consistent structural implication for residues D629, E728 and E729; they all lined inner vestibule of the channel. E648 is located at the connecting‐helix of the channel and faces the into cytoplasmic face. Hence we postulated that the residue may contribute to the inward‐rectification by reducing access resistance of spermine from channel periphery into inner vestibule. The location of residues fitted well with electrical distance.Support or Funding Information*This work was supported by the National Research Foundation of Korea, which is funded by the Ministry of Science, ICT (Information & Communication Technology), and Future Planning (MSIP) of the Korean Government (2018R1A41023822 to I. So) and by the Education and Research Encouragement Fund of Seoul National University Hospital (I. So). Christine Haewon Park was supported by the BK plus program from the MSIP.