PIP2 Interacts Electrostatically with MARCKS-like Protein-1 and ENaC in Renal Epithelial Cells

• Authors: Qiang Yue, Emory University; Otor Al-Khalili, Emory University; Auriel Moseley, Emory University; Masaaki Yoshigi, University of Utah; Brandi Wynne, Emory University; He-Ping Ma, Emory University; Douglas Eaton, Emory University
• Journal Title: BIOLOGY-BASEL
• Volume: Volume 11, Number 12
• Publisher: MDPI | 2022-12-01
• Type of Work: Article | Final Publisher PDF
• Publisher DOI: http://doi.org/10.3390/biology11121694
• Subjects: Health Sciences, Medicine and Surgery
• Keywords: Science & Technology; Life Sciences & Biomedicine; Biology; Life Sciences & Biomedicine - Other Topics; MARCKS-like protein-1; PIP2; MARCKS-like-1; ENaC; mpkCCD cells; SODIUM-CHANNEL ENAC; C-KINASE SUBSTRATE; COLLECTING DUCT; NA+ CHANNEL; PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE; DIRECT ACTIVATION; LIPID RAFTS; MEMBRANE; PI(4,5)P-2; 3,4,5-TRISPHOSPHATE

Abstract

We examined the interaction of a membrane-associated protein, MARCKS-like Protein-1 (MLP-1), and an ion channel, Epithelial Sodium Channel (ENaC), with the anionic lipid, phosphatidylinositol 4, 5-bisphosphate (PIP2). We found that PIP2 strongly activates ENaC in excised, inside-out patches with a half-activating concentration of 21 ± 1.17 µM. We have identified 2 PIP2 binding sites in the N-terminus of ENaC β and γ with a high concentration of basic residues. Normal channel activity requires MLP-1’s strongly positively charged effector domain to electrostatically sequester most of the membrane PIP2 and increase the local concentration of PIP2. Our previous data showed that ENaC covalently binds MLP-1 so PIP2 bound to MLP-1 would be near PIP2 binding sites on the cytosolic N terminal regions of ENaC. We have modified the charge structure of the PIP2 –binding domains of MLP-1 and ENaC and showed that the changes affect membrane localization and ENaC activity in a way consistent with electrostatic theory.