Schaffhauser DF et al. (2012), An integrated field-effect microdevice for moni...

XB-ART-45615

PLoS One 2012 Jan 01;77:e39238. doi: 10.1371/journal.pone.0039238.

Show Gene links Show Anatomy links

An integrated field-effect microdevice for monitoring membrane transport in Xenopus laevis oocytes via lateral proton diffusion.

Schaffhauser DF , Patti M , Goda T , Miyahara Y , Forster IC , Dittrich PS .

???displayArticle.abstract???
An integrated microdevice for measuring proton-dependent membrane activity at the surface of Xenopus laevis oocytes is presented. By establishing a stable contact between the oocyte vitelline membrane and an ion-sensitive field-effect (ISFET) sensor inside a microperfusion channel, changes in surface pH that are hypothesized to result from facilitated proton lateral diffusion along the membrane were detected. The solute diffusion barrier created between the sensor and the active membrane area allowed detection of surface proton concentration free from interference of solutes in bulk solution. The proposed sensor mechanism was verified by heterologously expressing membrane transport proteins and recording changes in surface pH during application of the specific substrates. Experiments conducted on two families of phosphate-sodium cotransporters (SLC20 & SLC34) demonstrated that it is possible to detect phosphate transport for both electrogenic and electroneutral isoforms and distinguish between transport of different phosphate species. Furthermore, the transport activity of the proton/amino acid cotransporter PAT1 assayed using conventional whole cell electrophysiology correlated well with changes in surface pH, confirming the ability of the system to detect activity proportional to expression level.

???displayArticle.pubmedLink??? 22792166
???displayArticle.pmcLink??? PMC3390327
???displayArticle.link??? PLoS One

Species referenced: Xenopus laevis
Genes referenced: cad cald1 slc20a2 slc6a1

???attribute.lit??? ???displayArticles.show???

	Figure 1. Lateral proton diffusion model.Simplified cartoon of the proton-selective lateral diffusion model where the oocyte interfaces to the sensor element (not to scale). The vitelline layer of the (defolliculated) oocyte comprises a network of fibrous filaments that surround the cytoplasmic membrane, which itself is not smooth due to numerous microvilli protrusions (not shown). The total thickness of the vitelline layer is estimated to be 2–3 µm for stage VI oocytes. [39] ([S]: concentration of substrate interacting with the membrane transport proteins. [H+]D, [H+]S, [H+]B: Proton concentration at the detection site, membrane surface and in buffer, respectively.) We assume the diffusion barrier is narrow compared with the sensor area so that “edge effects” can be neglected and transporters within the sensor region do not “see” the substrate S.
	Figure 2. Design of the microdevice.a) Schematic view of the cross-section of the device (not drawn to scale). The height of the microperfusion channel is around 200 µm. A typical Xenopus oocyte would be 1000–1200 µm in diameter. b) Micrograph of the sensor as seen through the hole (ca. 800 µm in diameter) of the oocyte immobilization compartment. The oocyte membrane completely covers the active area of the sensor due to its deformability. c) Exploded view of the device using the original 3D CAD engineering data. d) Photograph of the assembled, but unconnected device.
	Figure 3. pH reference measurements.a) Time-dependent measurement of phosphate reference buffers at pH 4.01, pH 7.00 and pH 9.21 and b) linear fit of the data points obtained from the baselines of each buffer. Slope and standard error of the linear fit are shown. c) Time-dependent measurement of 100 Na solutions buffered at pH 7.40 and pH 7.50 for two different perfusion pressures (100 mbar and 200 mbar). d) Expanded view of data in panel c during the exchange of solutions from pH 7.40 to pH 7.50. Valves were switched at t = 0 s.
	Figure 4. Membrane transport experiments.Experiments conducted on oocytes heterologously expressing various membrane transport proteins indicated with their respective controls on non-injected (NI) oocytes showing sensor readout (VSG) as a function of time. Only part of the initial stabilizing baseline region that preceded substrate application is shown (see Materials and Methods): a) PAT1, b) NaPi IIb, c) NaPi-IIc, d) PiT-2, e) Proline control, f) Pi control, g) GAT1, h) ENaC. In each case either the same or representative oocytes from the same batch were pretested using a two-electrode voltage clamp to confirm functional expression. The bars indicate the duration of application of the respective activating and blocking agents. Arrows indicate flux direction of substrate according to the assumed driving force conditions.
	Figure 5. Correlation of pH response with protein expression level.Correlating sensor response with transport activity. a) Sensor response to proline superfusion of a representative oocyte (designated #4 in c) heterologously expressing PAT1. b) TEVC I-V data of the proline-dependent current of oocyte #4 in response to the addition of 3 mM proline solution to the 100 Na buffer. Inset shows the change in membrane potential induced by proline application for the same oocyte as in a. c) Correlation of ΔVSG and the substrate-dependent current. Each point represents data from a single oocyte. Arrow marks the data point of oocyte #4 (−23 mV, −140 nA).

References [+] :

Antonenko, Coupling of proton source and sink via H+-migration along the membrane surface as revealed by double patch-clamp experiments. 1998, Pubmed