Potassium Channel-Based Two Component Optogenetic Tool For Silencing Of Excitable Cells

Opsin-based optogenetic tools still lack a light-gated potassium channel for cell inhibition. Silencing of excitable cells had been achieved so far by the use of light driven pumps for protons and chloride as well as light-gated chloride channels. Unfortunately, changes on pH or Cl- concentration could lead to undesirable effects on synaptic transmission limiting the use of these tools for neuronal silencing. We have developed a potassium channel-based inhibitory tool combining the small cAMP-gated potassium channel SthK (from Spirochaeta thermophile) and a photoactivated adenylyl cyclase. From all the PACs tested we found that bPAC (from the bacterium Beggiatoa), and TpPAC (from bacterium Turneriella parva) produced the best results. Characterization of both optogenetic-tools in ND7/23 cells showed that application of short blue light pulses to cells transfected with SthK and a PAC, produced high-amplitude potassium outward currents that are dependent of the light intensity. The TpPAC-based construct needed 10 times more light to reach the maximum current compared to bPAC. However, this is an advantage in biological systems where a lower production of cAMP is needed or where the ubiquitous phosphodiesterase activity is very low. Two construct configurations were made. In the first configuration the SthK channel and the PAC are expressed as a fused protein (PAC-SthK) allowing localized expression of cAMP at the plasma membrane. In the second configuration both proteins are separated by a P2A site (SthK-P2A-PAC) and the PAC is expressed in the cytoplasm. The fused constructs produced an increase on the current's latency and a decrease on the current density. Nevertheless, both configurations were capable of inhibiting neuronal action potential firing in cultures of hippocampal neurons. Cyclic nucleotide-gated potassium channel - photoactivated cyclese based constructs are a very promising tool for acute optogenetic inhibition.