Curated Optogenetic Publication Database

Abstract: The cardiac sodium channel NaV1.5 initiates each heartbeat by generating the rapid depolarizing upstroke of the action potential. Dysregulation of NaV1.5 gating can produce cardiac arrhythmias by slowing inactivation, increasing late sodium current (INa,L), and impairing electrical stability. Here, we show that phosphatidylinositol-4,5-bisphosphate (PIP2) is a critical membrane cofactor that stabilizes NaV1.5 gating. Acute PIP2 depletion in human iPSC-derived cardiomyocytes, produced by activation of endogenous AT1 receptors, activation of an engineered M3q-DREADD, or optogenetic recruitment of CRY2-pseudojanin, shifted voltage dependence, slowed fast inactivation, and increased INa,L. These effects were prevented by augmenting intracellular PIP2, required PLC activity when driven by Gq-coupled receptors, and were independent of downstream Ca2 or PKC signaling. Unlike the skeletal-muscle isoform NaV1.4, NaV1.5 displayed PIP2-dependent shifts in both activation and steady-state inactivation, indicating isoform-specific lipid coupling. Induced-fit docking and molecular dynamics simulations identified a PIP2-interaction interface between the domain IV voltage sensor and pore that contains disease-linked residues. The disease-reported variant R1644C weakened and redistributed the predicted PIP2-contact network, produced elevated basal INa,L, showed enhanced sensitivity to PIP2 depletion, and caused an approximately 30-fold reduction in apparent functional PIP2 sensitivity in excised patches. These findings define a lipid-dependent mechanism that stabilizes NaV1.5 gating and reveal how physiological Gq signaling and inherited channel variants can converge on the channel-PIP2 axis to promote proarrhythmic late sodium current.

Abstract: The Protein kinase C family consists of several closely related kinases. These enzymes regulate the function of proteins through the phosphorylation of hydroxyl groups on serines and/or threonines. The selective activation of individual PKC isozymes has proven challenging due to a lack of specific activator molecules. Here we developed an optogenetic, blue-light activated PKC isozyme that harnesses a plant-based dimerization system between the photosensitive cryptochrome-2 (CRY2) and the N-terminus of the transcription factor CIB1 (CIBN). We show that tagging CRY2 with the catalytic domain of PKC isozymes can efficiently promote its translocation to the cell surface upon blue light exposure. We demonstrate this system using PKCε and show that this leads to robust activation of a K+ channel (GIRK1/4) previously shown to be activated by PKCε. We anticipate that this approach can be utilized for other PKC isoforms to provide a reliable and direct stimulus for targeted membrane protein phosphorylation by the relevant PKCs.

Abstract: Transient receptor potential canonical type 5 (TRPC5) ion channels are expressed in the brain and kidney, and have been identified as promising therapeutic targets whose selective inhibition can protect against diseases driven by a leaky kidney filter, such as Focal Segmental Glomerular Sclerosis (FSGS). TRPC5 channels are activated by elevated levels of extracellular Ca2+or lanthanide ions, but also by G protein (Gq/11) stimulation. Phosphatidylinositol bisphosphate (PIP2) hydrolysis by phospholipase C (PLC) enzymes leads to protein kinase C (PKC)-mediated phosphorylation of TRPC5 channels and their subsequent desensitization. However, the roles of PIP2 in activation and maintenance of TRPC5 channel activity via its hydrolysis product diacyl glycerol (DAG), as well as the mechanism of desensitization of TRPC5 activity by DAG-stimulated PKC activity remain unclear. Here, we designed experiments to distinguish between the processes underlying channel activation and inhibition. Using whole-cell patch clamp, we employed an optogenetic tool to dephosphorylate PIP2 and assess channel-PIP2 interactions influenced by activators, such as DAG, or inhibitors, such as PKC phosphorylation. Using total internal reflection microscopy, we assessed channel cell surface density. We show that PIP2 controls both the PKC-mediated inhibition as well as the DAG- and lanthanide-mediated activation of TRPC5 currents via control of gating rather than channel cell surface density. These mechanistic insights promise to aid in the development of more selective and precise inhibitors to block TRPC5 channel activity, and to illuminate new opportunities for targeted therapies for a group of chronic kidney diseases for which there is currently a great unmet need.