An integrated field-effect microdevice for monitoring membrane transport in Xenopus laevis oocytes via lateral proton diffusion.
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.
Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland.
Daniel Felix
DF
Schaffhauser
Monica
M
Patti
Tatsuro
T
Goda
Yuji
Y
Miyahara
Ian Cameron
IC
Forster
Petra Stephanie
PS
Dittrich
D000818:Animals; D001692:Biological Transport; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism*; D018408:Patch-Clamp Techniques / Q000295:instrumentation / Q000379:methods; D011522:Protons; D014817:Vitelline Membrane / Q000378:metabolism*; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism*
D026901:Membrane Transport Proteins; D011522:Protons