Lipid and protein interactions at the C‐terminal part of TRPM4

Abstract

TRPM4 is a Ca2+‐impermeable Ca2+‐activated cation channel that undergoes a fast desensitization by intracellular Ca2+ ([Ca2+]i). The desensitization of TRPM4 to [Ca2+]i is attenuated by phosphatidylinositol (4,5)‐biphosphate (PIP2) and requires a pleckstrin homology‐like domain localized in the C terminus of the channel. Our data suggest that some of enzymes involved in the PIP2 metabolism, such as a phospholipase C, a PIP2‐regenerating kinase, and a PIP2‐depleting phosphatase might be functionally associated with TRPM4. To examine direct interaction of TRPM4 with phosphoinositides as well as formation of a signaling complex with other proteins, we fused the C‐terminal part of TRPM4 to GST and expressed in E. coli. The purified fusion protein was able to interact with membrane lipids in lipid‐protein overlay experiments. In order to identify interacting protein partners, we performed GST‐pulldown experiments with protein lysates from cells endogenously expressing TRPM4 channels. The mass spectrophotometric analysis revealed several putative interactions at the C terminus of TRPM4 that will be reconfirmed and further tested at a functional level. Supported by Interuniversity Poles of Attraction Program IUAP Nr.3P4/23, Excellentiefinanciering EF/95/010, Research Foundation‐Flanders (G.0565.07).