Impacts of the osmolality and the lumenal ionic strength on osmosensory transporter ProP in proteoliposomes

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.

FIGURE 1.

Properties of ProP, BetP, and OpuA (, , –49). In Part A, ABC is ATP Binding Cassette Transporter Family, BCCT is Betaine Carnitine Choline Transporter Family (BetP is also a member of the LeuT structure family), and MFS is Major Facilitator Superfamily. NT is not tested.

FIGURE 2.

Impacts of K⁺, Na⁺, and phosphate on ProP activity. PRLs were prepared in 0. 1 m potassium phosphate, 0.5 mm EDTA, 2 mm 2-mercaptoethanol, pH 7.4, and the lumenal buffer was modified as explained under “Experimental Procedures.” Δμ̃_H⁺ (protonmotive force) was imposed by diluting PRLs prepared in potassium phosphate, pH 7.4, 200-fold into sodium phosphate, pH 6.4, in the presence of valinomycin, and proline uptake activities were determined as a function of the osmolality using assay and wash buffers of equivalent sodium phosphate concentration (e.g. 10 mm sodium phosphate, pH 6.4, for PRLs loaded with 10 mm potassium phosphate, pH 7.4). The assay medium osmolality was adjusted with sorbitol. The data were fit to Equation 2 to obtain A_max and Π½/RT as previously described (10). A, PRLs were loaded with potassium phosphate at a series of concentrations (10–100 mm) so that K⁺ and phosphate concentrations varied in parallel. B, A_max and Π½/RT values derived by fitting data in panel A to Equation 2 are plotted versus the initial lumenal phosphate concentration of each preparation. C, PRLS were loaded with potassium phosphate/sodium phosphate mixtures so that the K⁺ and Na⁺ concentrations varied reciprocally, but the phosphate concentration remained constant at 100 mm. D, A_max and Π½/RT values derived by fitting data in panel C to Equation 2 are plotted versus the initial lumenal phosphate concentration contributed by potassium phosphate. E, data from panel A are replotted to show the relationship between the initial rate of proline uptake (a₀) and the lumenal phosphate concentration (calculated assuming that the PRLs behave as ideal osmometers). F, PRLs were loaded with 10 mm KPi alone (white circles) or 10 mm KPi plus KCl (160 mm, gray circles) or NaPi (90 mm, black circles).

FIGURE 3.

Variations in lumenal pH of liposomes and PRLs. Liposomes were prepared in potassium phosphate (100 mm (panel A) or 10 mm (panel B)), and the potassium phosphate content of PRLs was adjusted to 100 mm (panels C and E) or 10 mm (panels D and F). Liposomes and PRLs were loaded with pyranine (0.4 μm) as described under “Experimental Procedures.” These preparations were diluted 200-fold into sodium phosphate buffer of the same concentration at pH 6.4 (all panels) or 7.4 (panels A and B) as indicated, and pyranine fluorescence was monitored (excitation wavelength 460 nm, emission wavelength 510 nm). Valinomycin (V, 0.19 μm) with or without proline (P, 0.2 mm) and FCCP (F, 5 μm) were added subsequently as indicated.

FIGURE 4.

Dependence of ProP activity on the osmolality and the lumenal ionic strength of PRLs. Left panels. PRLs were loaded with potassium phosphate at the indicated concentration plus the K⁺ or Na⁺ salt of a listed anion to adjust the initial lumenal K⁺ concentration to 178 mm, and proline uptake rates were measured as outlined in the legend to Fig. 2. The assay and wash buffers were identical to the PRL loading buffers except that Na⁺ replaced K⁺, and sorbitol was used in place of NaI because I⁻ interfered with scintillation counting. These variations in ion composition did not affect the membrane potential as indicated by 3,3′-dipropylthiadicarbocyanine iodide fluorescence (data not shown). The assay medium osmolality was adjusted with sorbitol. The data were fit to Equation 2, with X as the osmolality or the lumenal ionic strength, the latter calculated with Equation 3. Estimates of the osmolality (Π½/RT) and the ionic strength (I_1/2) at which ProP activity was half-maximal are shown. The primary data are provided in supplemental Fig. S2. Right panels, the ionic strength of the PRL lumen attained at each (measured) osmolality during the titrations outlined above was calculated with the assumption that the PRLs behaved as ideal osmometers.

FIGURE 5.

The impacts of PEG1000 and BSA on ProP activity in PRLs. A and B, PRLs were prepared in 0.1 m potassium phosphate and loaded with nothing (circles) or with PEG1000 (triangles, 83 g/liter). Initial rates of proline uptake were measured as described under “Experimental Procedures.” A, Relative proline uptake rates, determined as a function of the osmolality, were derived from Fig. 5 of Culham et al. (10). They are replotted here as a basis for further analyses. The A_max values were 1.6 and 2.1 μmol/min/mg ProP protein for unloaded and PEG1000-loaded PRLs, respectively. B, relative proline uptake rates were plotted versus the lumenal ionic strength, calculated with the assumption that the PRLs acted as ideal osmometers (open symbols) or corrected to account for the non-ideality of potassium phosphate-PEG1000 mixtures illustrated in panel E (solid triangles, see “Experimental Procedures”). C and D, PRLs were prepared in 0.1 m potassium phosphate and loaded with nothing (circles) or with BSA (triangles, 181 g/liter). Initial rates of proline uptake were measured as described under “Experimental Procedures.” C, relative proline uptake rates are plotted as a function of the osmolality, which was adjusted with NaCl (open symbols) or glucose (closed symbols). The A_max values were 0.9 and 1.1 μmol/min/mg ProP protein for unloaded PRLs and BSA-loaded PRLs, respectively. D, relative proline uptake rates are plotted versus the lumenal ionic strength, calculated with the assumption that the PRLs acted as ideal osmometers. E and F, solutions of potassium phosphate (KPi), KP_i plus PEG1000, and KP_i plus BSA were prepared to simulate the conditions in our osmotically shrunken PRLs then diluted to simulate the range of concentrations that would occur during an osmolality titration (see “Experimental Procedures” and the legend to supplemental Fig. S1). Corresponding solutions containing only KP_i, PEG1000, or BSA were also prepared. The osmolalities of the solutions were measured and plotted versus the corresponding KP_i or equivalent PEG1000 (E) or BSA (F) concentration. The data in panel E were used as explained under “Experimental Procedures” to correct the relationship between relative proline uptake rate and lumenal ionic strength illustrated in panel B.

FIGURE 6.

Liposomes can be loaded with BSA by extrusion. Liposomes were prepared and unloaded (Nil) or loaded with glucose, PEG1000, or BSA, and the intensity and intensity fluctuations of light scattered at 90° to the incident beam were analyzed by dynamic light-scattering spectrometry (DLS) as described under “Experimental Procedures.” A, shown are the properties of the solvents with which the liposomes were loaded and the sizes and scattering intensities of the unloaded and loaded liposomes. B, the size distributions of unloaded and BSA-loaded liposomes was determined by dynamic light-scattering spectroscopy.