Role of the Scaffold Protein MIM in the Actin-Dependent Regulation of Epithelial Sodium Channels (ENaC)

Abstract

Epithelial Sodium Channels (ENaCs) are expressed in different organs and tissues, particularly in the cortical collecting duct (CCD) in the kidney, where they fine tune sodium reabsorption. Dynamic rearrangements of the cytoskeleton are one of the common mechanisms of ENaC activity regulation. In our previous studies, we showed that the actin-binding proteins cortactin and Arp2/3 complex are involved in the cytoskeleton-dependent regulation of ENaC and that their cooperative work decreases a channel's probability of remaining open; however, the specific mechanism of interaction between actin-binding proteins and ENaC is unclear. In this study, we propose a new component for the protein machinery involved in the regulation of ENaC, the missing-in-metastasis (MIM) protein. The MIM protein contains an IMD domain (for interaction with PIP₂ -rich plasma membrane regions and Rac GTPases; this domain also possesses F-actin bundling activity), a PRD domain (for interaction with cortactin), and a WH2 domain (interaction with G-actin). The patch-clamp electrophysiological technique in whole-cell configuration was used to test the involvement of MIM in the actin-dependent regulation of ENaC. Co-transfection of ENaC subunits with the wild-type MIM protein (or its mutant forms) caused a significant reduction in ENaC-mediated integral ion currents. The analysis of the F-actin structure after the transfection of MIM plasmids showed the important role played by the domains PRD and WH2 of the MIM protein in cytoskeletal rearrangements. These results suggest that the MIM protein may be a part of the complex of actin-binding proteins which is responsible for the actin-dependent regulation of ENaC in the CCD.

Keywords: Arp2/3 complex; ENaC; MIM; cortactin; cytoskeleton.

Fig. 1

Domain structure of the mouse MIM protein (encoded by mtss1, UniProt Q8R1S4). The IMD domain can bind to F-actin, PIP2 rich membrane areas and Rac GTPases, and also plays an essential role in the dimerization of MIM. The SRD domain contains sites of Tyr phosphorylation. The PRD domain interacts with cortactin and tyrosine phosphatase delta. The WH2 domain binds G-actin

Fig. 2

Actin cytoskeleton arrangement after transfection with different types of the MIM protein. Images of the actin cytoskeleton acquired with a confocal microscope (typical micrographs from 3 independent experiments) in fixed CHO cells after transient transfection with plasmids encoding different forms of the MIM protein (each plasmid based on pEGFP vector). GFP – control transfection; MIM full – full-length protein; MIM PH – chimeric protein, where the inactive IMD domain is conjugated with the PH domain of PLCD1, which leads to MIM’s inability to dimerize); MIM ΔPRD – the PRD domain (Δ617-727) of MIM is removed, and the protein cannot interact with cortactin; MIM ΔWH2 – the WH2 domain (Δ746-759) of MIM is removed, and this form of MIM cannot polymerize G-actin; MIM/IMD-L – an isolated long splice variant of the IMD domain (the rest of the MIM protein is absent), which cannot interact with Rac GTPases. I –rhodamine-phalloidine emission (red). II – magnified images of selected areas: upper panel – rhodamine-phalloidine emission, lower panel – merged image. III – merged image of GFP (green), rhodamine-phalloidine (red) and Hoechst-33342 (nuclear acids dye, blue) emissions

Fig. 3

Effect of different forms of MIM on the amiloride-sensitive ENaC current density. A – summarized histogram of amiloride-sensitive current densities taken from electrophysiological experiments (patch-clamp in whole-cell configuration). CHO cells were co-transfected with mENaC plasmids, together with GFP (control), or mENaC with different types of the MIM protein (n – number of independent experiments; * – p < 0.05) B – representative traces of typical ENaC-mediated integral currents (ENaC – current magnitude, A – amiloride application at the end of the experiment (10 μM)). C – Schematic illustration of the experimental protocol

Fig. 4

Suggested scheme of actin-dependent regulation of ENaC by the actin-binding proteins MIM, cortactin, and the Arp2/3 complex