Evaluating methods and protocols of ferritin-based magnetogenetics.

FeRIC (Ferritin iron Redistribution to Ion Channels) is a magnetogenetic technique that uses radiofrequency (RF) alternating magnetic fields to activate the transient receptor potential channels, TRPV1 and TRPV4, coupled to cellular ferritins. In cells expressing ferritin-tagged TRPV, RF stimulation increases the cytosolic Ca2+ levels via a biochemical pathway. The interaction between RF and ferritin increases the free cytosolic iron levels that, in turn, trigger chemical reactions producing reactive oxygen species and oxidized lipids that activate the ferritin-tagged TRPV. In this pathway, it is expected that experimental factors that disturb the ferritin expression, the ferritin iron load, the TRPV functional expression, or the cellular redox state will impact the efficiency of RF in activating ferritin-tagged TRPV. Here, we examined several experimental factors that either enhance or abolish the RF control of ferritin-tagged TRPV. The findings may help optimize and establish reproducible magnetogenetic protocols.

A Comprehensive Optogenetic Pharmacology Toolkit for In Vivo Control of GABA(A) Receptors and Synaptic Inhibition.

Exogenously expressed opsins are valuable tools for optogenetic control of neurons in circuits. A deeper understanding of neural function can be gained by bringing control to endogenous neurotransmitter receptors that mediate synaptic transmission. Here we introduce a comprehensive optogenetic toolkit for controlling GABA(A) receptor-mediated inhibition in the brain. We developed a series of photoswitch ligands and the complementary genetically modified GABA(A) receptor subunits. By conjugating the two components, we generated light-sensitive versions of the entire GABA(A) receptor family. We validated these light-sensitive receptors for applications across a broad range of spatial scales, from subcellular receptor mapping to in vivo photo-control of visual responses in the cerebral cortex. Finally, we generated a knockin mouse in which the "photoswitch-ready" version of a GABA(A) receptor subunit genomically replaces its wild-type counterpart, ensuring normal receptor expression. This optogenetic pharmacology toolkit allows scalable interrogation of endogenous GABA(A) receptor function with high spatial, temporal, and biochemical precision.

Tyrosine phosphorylation of rod cyclic nucleotide-gated channels switches off Ca2+/calmodulin inhibition.

Cyclic nucleotide-gated (CNG) ion channels are crucial for phototransduction in rod photoreceptors. Light triggers a biochemical cascade that reduces the concentration of cGMP in rods, closing CNG channels, which leads to membrane potential hyperpolarization and a decrease in the concentration of intracellular Ca2+. During light adaptation, the sensitivity of CNG channels to cGMP is decreased by Ca2+, which in conjunction with calmodulin (CaM), binds directly to CNG channels. The cGMP sensitivity of rod CNG channels is also reduced by phosphorylation of specific tyrosine residues in the three CNGA1 subunits and one CNGB1 subunit that comprise the rod channel. Here we show that phosphorylation prevents Ca2+/CaM inhibition. Experiments on native channels in rod outer segments and expressed channels in Xenopus oocytes show that Ca2+/CaM inhibition can be toggled off or on by promoting phosphorylation or dephosphorylation, respectively. Experiments in which the crucial tyrosine phosphorylation sites in CNGA1 and CNGB1 are replaced with phenylalanines show that residue Y498 in CNGA1 is the phosphorylation site responsible for regulating Ca2+/CaM inhibition. Ca2+/CaM inhibits the rod channel by binding to the N terminus of the CNGB1 subunit, causing it to uncouple from the C terminus of CNGA1. We propose that phosphorylation of CNGA1Y498, on the C terminus of CNGA1, triggers an equivalent uncoupling from the C terminus of CNGB1, thereby curtailing Ca2+/CaM inhibition. The control of CaM inhibition by CNG channel phosphorylation may be important for light adaptation and the regulation of phototransduction by IGF-1, a retinal paracrine factor that alters the tyrosine phosphorylation state of rod CNG channels.

Subunit contributions to phosphorylation-dependent modulation of bovine rod cyclic nucleotide-gated channels.

Cyclic nucleotide-gated (CNG) channels in rod photoreceptors transduce a decrease in cGMP into hyperpolarization during the light response. Insulin-like growth factor-1 (IGF-1) increases light responses by increasing the cGMP sensitivity of CNG channels, an event mediated by a protein tyrosine phosphatase. Native rod CNG channels are heteromultimers, composed of three CNGA1 subunits and one CNGB1 subunit. Previous studies on heterologously expressed rod CNG channels show that a specific tyrosine in the CNGA1 subunit (Y498) is required for modulation by protein tyrosine phosphatases, protein tyrosine kinases and IGF-1. Here we show that the CNGB1 subunit contains a specific tyrosine (Y1097) that is important for modulation of heteromeric channels by tyrosine phosphorylation. Direct biochemical measurements demonstrate 32P-labelling of CNGA1Y498 and CNGB1Y1097. Replacement of either Y498 of CNGA1 or Y1097 of CNGB1 with phenylalanine reduces modulation, and removal of both tyrosines eliminates modulation. Unlike CNGA1, CNGB1 does not exhibit activity dependence of modulation by tyrosine phosphorylation. Hence both CNGA1 and CNGB1 subunits contribute to phosphorylation-dependent modulation of rod CNG channels, but the phosphorylation states of the two subunits are regulated in different ways.