Water channel pore size determines exclusion properties but not solute selectivity

Philip Kitchen,Alex C Conner,Susanna Törnroth-Horsefield,Simone U Pickel,Mootaz M Salman,Matthew T Conner,Roslyn M Bill,Jordan Jennings

doi:10.1038/s41598-019-56814-z

Abstract

Aquaporins (AQPs) are a ubiquitous family of transmembrane water channel proteins. A subgroup of AQP water channels also facilitates transmembrane diffusion of small, polar solutes. A constriction within the pore, the aromatic/arginine (ar/R) selectivity filter, is thought to control solute permeability: previous studies on single representative water channel proteins suggest narrow channels conduct water, whilst wider channels permit passage of solutes. To assess this model of selectivity, we used mutagenesis, permeability measurements and in silico comparisons of water-specific as well as glycerol-permeable human AQPs. Our studies show that single amino acid substitutions in the selectivity filters of AQP1, AQP4 and AQP3 differentially affect glycerol and urea permeability in an AQP-specific manner. Comparison between in silico-calculated channel cross-sectional areas and in vitro permeability measurements suggests that selectivity filter cross-sectional area predicts urea but not glycerol permeability. Our data show that substrate discrimination in water channels depends on a complex interplay between the solute, pore size, and polarity, and that using single water channel proteins as representative models has led to an underestimation of this complexity.

Highlights

Aquaporins (AQPs) are a ubiquitous family of transmembrane water channel proteins
For large protein families with high sequence, structural and functional homologies, the characteristics of model proteins may often be generalised to the whole family
In the case of the highly-homologous family of water channels, the proposed mechanism for solute selectivity is largely derived from comparing structural details of the water-specific channel AQP1 to those of GlpF from E. coli, which is permeable to larger solutes such as glycerol and urea

Summary

Introduction

Aquaporins (AQPs) are a ubiquitous family of transmembrane water channel proteins. A subgroup of AQP water channels facilitates transmembrane diffusion of small, polar solutes. NPA motifs are not thought to contribute directly to the selectivity differences between wAQPs and GLPs, subtle structural differences affecting this conserved motif may partially account for quantitative differences in single-channel water permeability between different AQPs. The second AQP region involved in selectivity, the ar/R-motif, is located towards the extracellular side of the pore and is responsible for determining the difference in solute permeability between wAQPs and GLPs, as well as playing a role in proton exclusion. The second AQP region involved in selectivity, the ar/R-motif, is located towards the extracellular side of the pore and is responsible for determining the difference in solute permeability between wAQPs and GLPs, as well as playing a role in proton exclusion It is formed by four amino acid residues from disparate locations in the primary sequence (Fig. 1B,C), of which the arginine in position 4 is highly conserved throughout the AQP family. Functional studies of H+ permeability in superaquaporins are yet to be reported, the loss of this arginine residue may suggest roles in intracellular H+ homeostasis for AQPs 11 and 12

Methods

Results

Conclusion