Engineering K+channels for Long Term Inhibition

We are using different strategies for modulating potassium (K⁺) channel activity by light. One strategy relies on the engineering of a fully genetically-encoded light regulated K⁺channel (BLINK), obtained by fusing LOV2 domain to the small viral channel Kcv. We have optimized this system in such a way that it expresses in neurons and allows long lasting light-off activity and, in turn, long lasting inhibition of neurons. A second approach is based on light-dependent modulation of native HCN channels. These channels that control pacemaking activity in brain and heart, have a regulatory subunit TRIP8b that prevents cAMP induced enhancement of their current. We have reduced TRIP8b to a short peptide, nanoTRIP, and fused it to a LOV2 domain that releases the active peptide upon blue light. A final strategy uses a light-dependent dimerization system to activate the transcription of small K⁺channels. With this system we can target active K+ channels not only to the plasma membrane but also to the inner mithochondrial membrane. This approach allows long lasting depolarization of the mitochondria with physiological consequences, such as modulation of mitochondrial calcium content.