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Micropipette Aspiration‐Based Assessment of Single Channel Water Permeability
Author(s) -
Boytsov Danila,
Hannesschlaeger Christof,
Horner Andreas,
Siligan Christine,
Pohl Peter
Publication year - 2020
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201900450
Subject(s) - vesicle , biophysics , pipette , chemistry , membrane , chromatography , permeability (electromagnetism) , fluorescence , analytical chemistry (journal) , biochemistry , biology , physics , quantum mechanics
Measurements of the unitary hydraulic conductivity of membrane channels, p f , may be hampered by difficulties in producing sufficient quantities of purified and reconstituted proteins. Low yield expression, the purely empiric choice of detergents, as well as protein aggregation and misfolding during reconstitution may result in an average of less than one reconstituted channel per large unilamellar vesicle. This limits their applicability for p f measurements, independent of whether light scattering or fluorescence quenching of encapsulated dyes is monitored. Here the micropipette aspiration technique is adopted because its superb sensitivity allows resolving p f values for one order of magnitude smaller protein densities in sphingomyelin and cholesterol rich giant unilamellar vesicles (GUVs). Protein density is derived from intensity fluctuations that fluorescently labeled channels in the aspirated GUV induce by diffusing through the diffraction limited spot. A perfusion system minimizes unstirred layers in the immediate membrane vicinity as demonstrated by the distribution of both encapsulated and extravesicular aqueous dyes. p f amounted to 2.4 ± 0.1 × 10 −13 cm³ s −1 for aquaporin‐1 that served as a test case. The new assay paves the way for directly monitoring the effect that interaction of aquaporins with other proteins or inhibitors may have on p f on a single sample.