Ionovation GmbH


	Concurrent optical and electrophysiological recordings of PorB


	Using the Ionovation Bilayer Explorer we determined the diffusion constant and the molecular brightness of PorB from Neisseria gonorrhoeae in a lipid bilayer and in the surrounding buffer while simultaneously observing channel activities under a controlled membrane potential. The diffusion constant was used to determine the oligomeric state of electrically active PorB in the bilayer.


	High definition data from a single eperiment

High resolution imaging

(a)

Bilayer set-up in the Ionovation Bilayer Slide

(b)

Fluoresently labeled PorB is perfused into the Trans-compartment and accumulates in the membrane.

(c)

Excess PorB was washed out using the automated perfusion unit of the Ionovation Bilayer Explorer.

Accumulation of PorB in the bilayer is quantified in a2 and c2.

High content electrophysiology

After addition of PorB to the Trans-compartment - above image (b) - multiple channel incorporations were detected electrically verifying the functionality of PorB.

Single molecule analysis

Left:

Fluorescence Intensity Distribution Analysis (FIDA)
The brightness of PorB increased ~3fold after incorporation into the bilayer. Fit 1 and 2 correspondent to the respective numbers in the images.

Right:

Fluorescence Correlation Spectroscopy (FCS)
Diffusion coefficient of PorB in solution was calculated as D(buff.) = 31µm²/s and as D(mem.) = 4.1µm²/s in the bilayer.

FIDA data directly and hydrodynamic calculations with the results derived from FCS experiments support the homotrimeric structure of the membrane-bound PorB and give a trimer radius of approx. 4 nm. With the innovative concept of the Ionovation Bilayer Explorer, the electrophysiological activity can directly be correlated to this oligomeric state of PorB.