The Significance and Mechanisms of Clustering by SLC4 Cotransporters in the Plasma Membrane

Abstract

The Solute Carrier 4 (SLC4) family are integral membrane cotransporters that reabsorb or secret anions across the basolateral membranes throughout the body, especially the kidney, eye, and red blood cells. Defects of SLC4A4 (NBCe1-A) lead to type-II acidosis and myriad ocular abnormalities; those of SLC4A1 (AE1) causes type-I acidosis and sperocytosis. We demonstrate that SLC4s form patches in the membrane of proximal tubule cells, corneal endothelial cells, and Xenopus oocytes. This new discovery of homophilic interactions or self-associations was initially suggested from our biophysical studies of the N-terminal domain of NBCe1-A (Nt) in solution, and by others studying the transmembrane domain (TMD) of AE1. The role and mechanisms of self-associations are further investigated, with insight into the role of the devastating R298S hereditary mutation found in type-II RTA afflicted individuals. We use confocal-microscopy imaging of fluorescently-tagged NBCe1-A and NBCe1-A-R298S molecules expressed in the aforementioned cells types and from in-depth biophysical studies of their Nts, including our Nt crystal structure at 2.4-Å resolution, melting-temperature, and homodimer dissociation constant (KD) analyses by composition-gradient multi-angle light scattering (CG-MALS). The structure reveals that the Nt dimerizes by two interlocking arms and contains two variable sequence regions, one at the extreme N terminus (V1) and another in the middle (V2). The V1 domains are flexible segments that gate substrate entry into the Nt, and that the V2 are large solvent accessible loops that extend ∼20 Å from the core Nt structure. Truncation mutants were rationally generated, and surface plasmon resonance (SPR) was used to investigate their roles in self-association. Based on these findings, we discuss the role of clustering in HCO3- sensing and transport in health and provide insight to the pathogenic processes observed in patients with point mutations.