An optical method for recording the activity of single transporters in membrane patches

Abstract

We have previously introduced an optical technique for recording the transport of fluorescent substrates by single membrane transporters. Referred to as optical single‐transporter recording (OSTR), the method was restricted to cases in which membrane transporters occurred at extremely small densities, namely at one or a few transporters per cell. Here we describe the extension of OSCR from whole cells harbouring a small number of transporters to small membrane patches containing transporters at normal densities. A technique was developed for firmly attaching cells to isoporous filters, i.e. very thin transparent sheets containing homogeneous populations of cylindrical pores. The flux of fluorescent transport substrates across the tiny membrane pieces spanning the filter pores was measured by scanning microphotolysis, a combination of fluorescence microphotolysis and confocal laser scanning microscopy. The technique was tested by attaching erythrocytes to filters containing pores of 1.2, 2.0 or 3.0 μm diameter. After treating filter‐attached erythrocyte membranes with streptolysin O, the transport of the fluorescent protein B‐phycoerythrin through single streptolysin O pores was observed. From the flux data the functional radius of the streptolysin O pore was derived to be 12.5 ± 0.9 nm, in very good agreement with previous electron microscopic estimates. The new technique features a number of unique properties: (i) the size of the membrane patch can be chosen within wide limits according to transporter density, (ii) transport can be recorded on many membrane patches in parallel, (iii) both influx and efflux may be analysed employing either photobleaching of fluorescent or photorelease of caged nonfluorescent substrates, (iv) two or more transport substrates may be monitored simultaneously. The new technique can be used, for instance, for analysing the activity of protein/particle pumps, a membrane transport domain not previously accessible to a single‐transporter analysis.

Optical recording of signal-mediated protein transport through single nuclear pore complexes

“…. transporters (5,7,40). The decomposition into subpopulations by Gaussians (lines) is shown in Fig.…”

“…Also, the sensitivity of the electrical patch clamp method requires a flow of Ͼ10 5 charges͞s to yield a detectable signal and therefore is most successfully applied to ion channels. We recently have established a method combining fluorescence microphotolysis (3) with confocal scanning microscopy (4) and a novel membrane patching method (5). This optical single-transporter recording (OSTR) is specific and sensitive enough to be applied to any membrane transport system including carriers and pumps provided the transport substrate is fluorescent or can be monitored via a fluorescent indicator.…”

Arrayed lipid bilayer chambers allow single-molecule analysis of membrane transporter activity

“…In an attempt to analyse transporter activity at the single-molecule level using optical methods, microsystems for micron-sized reactors sealed with lipid bilayers (lipid bilayer chambers) have been developed where transport is measured based on substrate accumulation in the chamber456. Although these methods have improved detection, single-molecule analysis of active transporters has not yet been achieved due to the technical difficulties of forming bilayer chambers with high reproducibility in a high-throughput manner.…”

Stable, Non‐Destructive Immobilization of Native Nuclear Membranes to Micro‐Structured PDMS for Single‐Molecule Force Spectroscopy

“…[12,13] In case of NE membranes lattice-structured surfaces using modified isoporous filters have been used previously to investigate the translocation-process through the NPC. [14] In this study, the NEs were investigated by optical single transporter recording (OSTR), [15] so as to measure passive transport through the NPCs' diffusion channels. In another study, Stoffler et al [16] used modified EM grids to avoid sample deformation for the investigation of calciummediated structural changes of the NPC.…”