Design of optoelectrodes for the remote imaging of cells and in situ electrochemical detection of neurosecretory events.

Optical fibers have opened avenues for remote imaging, bioanalyses and recently optogenetics. Besides, miniaturized electrochemical sensors have offered new opportunities in sensing directly redox neurotransmitters. The combination of both optical and electrochemical approaches was usually performed on the platform of microscopes or within microsystems. In this work, we developed optoelectrodes which features merge the advantages of both optical fibers and microelectrodes. Optical fiber bundles were modified at one of their extremity by a transparent ITO deposit. The electrochemical responses of these ITO-modified bundles were characterized for the detection of dopamine, epinephrine and norepinephrine. The analytical performances of the optoelectrodes were equivalent to the ones reported for carbon microelectrodes. The remote imaging of model neurosecretory PC12 cells by optoelectrodes was performed upon cell-staining with common fluorescent dyes: acridine orange and calcein-AM. An optoelectrode placed by micromanipulation at a few micrometers-distance from the cells offered remote images with single cell resolution. Finally, in situ electrochemical sensing was demonstrated by additions of K+-secretagogue solutions near PC12 cells under observation, leading to exocytotic events detected as amperometric spikes at the ITO surface. Such dual sensors should pave the way for in vivo remote imaging, optogenetic stimulation, and simultaneous detection of neurosecretory activities.

Unexpected Gating Behaviour of an Engineered Potassium Channel Kir.

In this study, we investigated the dynamics and functional characteristics of the KirBac3.1 S129R, a mutated bacterial potassium channel for which the inner pore-lining helix (TM2) was engineered so that the bundle crossing is trapped in an open conformation. The structure of this channel has been previously determined at high atomic resolution. We explored the dynamical characteristics of this open state channel using an in silico method MDeNM that combines molecular dynamics simulations and normal modes. We captured the global and local motions at the mutation level and compared these data with HDX-MS experiments. MDeNM provided also an estimation of the probability of the different opening states that are in agreement with our electrophysiological experiments. In the S129R mutant, the Arg129 mutation releases the two constriction points in the channel that existed in the wild type but interestingly creates another restriction point.