The microtubule‐associated End Binding proteins regulate the ER exporting and trafficking of the TRPM4 channels

Abstract

Trafficking and localization of ion channels are critical events for their proper function. These processes are relevant in several cellular functions, such as developmental programmes, cell polarity and local membrane potential. TRPM4 is a Ca2+‐activated non‐selective cationic channel expressed in different tissues and cell types, and is involved in a wide variety of physiological processes, such as insulin secretion and mechanotransduction. Altered activity, expression and localization of these channels might trigger several pathophysiological conditions. Indeed, TRPM4 plasma membrane overexpression has been related to cardiovascular and neurodegenerative diseases and cancer. Therefore, the mechanisms involved in the regulation of TRPM4 activity constitute an important area of biomedical research. Protein‐Protein Interactions (PPIs) control the expression, trafficking, localization and biophysical properties of ion channels. Thus, the identification of novel TRPM4‐related PPIs and their characterization might contribute to dissect the regulatory mechanisms of this channel. Interestingly, bioinformatics analyses of the primary sequence of TRPM4 allowed us to identify a putative interacting motif to End Binding (EB) proteins, novel members of the microtubule plus‐end tracking proteins (+TIPs). These proteins bind a consensus motif (SxIP) in their substrates and are involved in a plethora of cellular processes, including growing dynamics of the microtubule cytoskeleton, focal adhesion dynamics, cell migration and protein trafficking, targeting and localization. Here, we provide novel data suggesting that TRPM4 interacts with EB proteins. Moreover, we show that the mutation (TRPM4ΔSWIP and TRPM4SWNN) of the putative EB‐binding motif abolishes the TRPM4‐EB interaction. We also found that these mutant variants show a reduced expression of the mature population of the channel and display an endoplasmic reticulum (ER)‐like distribution. Furthermore, these mutant variants present a decreased expression in the plasma membrane, consistent with a reduction in the whole‐cell electrophysiological recordings. Conversely, we demonstrated that the expression of a soluble fragment containing the wild type N‐terminal region of TRPM4 (EGFP‐N‐TRPM4WT) reduces the membrane expression of TRPM4. These results suggest that EB interaction is necessary for the proper trafficking and activity of TRPM4. These findings might contribute to the understanding and characterization of novel mechanisms involved in TRP channel trafficking/localization, and could be applied to the design of therapeutic strategies against TRPM4 gain‐of‐function‐associated pathologies.Support or Funding InformationThis research was funded by Fondecyt 11121239 to O.C. and Fondecyt 11140064 to M.C. A.R. is a Conicyt Doctoral Fellowship recipient.