Impacts of the Osmolality and the Lumenal Ionic Strength on Osmosensory Transporter ProP in Proteoliposomes

Doreen E Culham,Michael Meinecke,Janet M Wood

doi:10.1074/jbc.m112.387936

Abstract

H(+) symporter ProP serves as a paradigm for the study of osmosensing. ProP attains the same activity at the same osmolality when the medium outside cells or proteoliposomes is supplemented with diverse, membrane-impermeant solutes. The osmosensory mechanism of ProP has been probed by varying the solvent within membrane vesicles and proteoliposomes. ProP activation was not ion specific, did not require K(+), and could be elicited by large, uncharged solutes polyethylene glycols (PEGS). We hypothesized that ProP is an ionic strength sensor and lumenal macromolecules activate ProP by altering ion activities. The attainable range of lumenal ionic strength was expanded by lowering the phosphate concentration within proteoliposomes. ProP activity at high osmolality, but not the osmolality, yielding half-maximal activity (Π(1/2)/RT), decreased with the lumenal phosphate concentration. This was attributed to acidification of the proteoliposome lumen due to H(+)-proline symport. The ionic strength yielding half-maximal ProP activity was more anion-dependent than Π(1/2)/RT for proteoliposomes loaded with citrate, sulfate, phosphate, chloride, or iodide. The anion effects followed the Hofmeister series. Lumenal bovine serum albumin (BSA) lowered the lumenal ionic strength at which ProP became active. Osmolality measurements documented the non-idealities of solutions including potassium phosphate and other solutes. The impacts of PEGS and BSA on ion activities did not account for their impacts on ProP activity. The effects of the tested solutes on ProP appear to be non-coulombic in nature. They may arise from effects of preferential interactions and macromolecular crowding on the membrane or on ProP.

Highlights

Transporter ProP serves as a paradigm for the study of osmosensing
In this report we extend that work by testing the hypothesis that ProP activity in PRLs is determined by the lumenal ionic strength
The lumenal concentrations of individual solutes and the lumenal ionic strength can be calculated on that basis, and the ionic strength at half-maximal ProP activity can be determined using Equation 2, with X ϭ I and X1⁄2 ϭ I1/2

Summary

Background

Transporter ProP serves as a paradigm for the study of osmosensing. Results: ProP activity does not correlate better with the lumenal ionic strength than the osmolality in proteoliposomes. ProP attains the same activity at the same osmolality when the medium outside cells or proteoliposomes is supplemented with diverse, membrane-impermeant solutes. Bacterial transporters ProP, OpuA, and BetP are paradigms for the study of osmosensing [1] They represent diverse evolutionary families, energy coupling mechanisms, and substrate specificities (Fig. 1). Their activities are sigmoid functions of the osmolality (⌸/RT) in cells and, after purification and reconstitution, in proteoliposomes (PRLs). In the previous study a higher osmolality was required to activate ProP in PRLs loaded with glucose or a membrane-impermeant PEG than those containing only buffer [10]. ProP activity correlated with the lumenal potassium phosphate concentration when the lumenal solvent included glucose or a small PEG (degree of polymerization up to 13). We show that lumenal bovine serum albumin (BSA) can elevate ProP activity and test the hypothesis that macromolecules alter the impact of potassium phosphate on ProP activity by altering ion activities

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION