Extrinsic control of single neurons and neuronal populations is a powerful approach for understanding how neural circuits function. Adding new thermogenetic tools to existing optogenetic and other forms of intervention will increase the complexity of questions that can be addressed. A good candidate for developing new thermogenetic tools is the Drosophila gustatory receptor family, which has been implicated in high-temperature avoidance behavior. We examined the five members of the Gr28b gene cluster for temperature-dependent properties via three approaches: biophysical characterization in Xenopus oocytes, functional calcium imaging in Drosophila motor neurons, and behavioral assays in adult Drosophila. Our results show that Gr28bD expression in Xenopus oocytes produces a non-specific cationic current that is activated by elevated temperatures. This current is non-inactivating and non-voltage dependent. When expressed in Drosophila motor neurons, Gr28bD can be used to change the firing pattern of individual cells in a temperature-dependent fashion. Finally, we show that pan-neuronal or motor neuron expression of Gr28bD can be used to alter fruit fly behavior with elevated temperatures. Together, these results validate the potential of the Gr28bD gene as a founding member of a new class of thermogenetic tools.

In fluorescence spectroscopy and imaging experiments with the fluorophores laurdan and 1,6-diphenyl-1,3,5-hexatriene (DPH) we found that phenol derivatives modulate membrane properties. We propose that activation of TRPA1 by non-electrophilic compounds may arise from the induction of changes in membrane properties and suggest that chemosensation may result from primary mechanosensory mechanisms.

and Gem/Kir (RGK) sub-family of small GTP-binding protein include e the most potent endogenous inhibitor of High-Voltage Activated (HVA) calcium channels. RGK proteins use several mechanisms to inhibit calcium current (I CA ): i) they promote dynamin-dependent endocytocys; ii) the lower the Po of the channel: ii) and they voltage sensor or reduction of charge. All RGK proteins associate directly with Ca 2þ channel b subunits (Cavb), and the binding between CaVa1 / Cavb has been shown to be essential for their inhibitory action for CaV1.2 and Ca V 2.1 and Ca V 2.2. In this study, we investigated inhibition of Ca V 2.3 Ca 2þ channels by RGK proteins. We found that when Xenopus laevis oocytes expressing Cav2.3 channels were injected with purified Gem protein, but not Rem, calcium currents where significantly decreased. This reduction was accompanied by a right shift in the conductance-voltage (GV curve) relationship of the channel. Furthermore, Gem also decreases the number of channels in the plasma membrane, evidenced by a reduction in maximal charge movement after injection of purified protein. The kinetic and voltage dependence of the reduced charge movement was not affected and thus immobilization of a subset of voltage-sensor seems unlikely. Surprisingly both effects are not dependent on the binding between CaVa1 to Cavb since CaV2.3 were expressed alone. Thus unlike than other neuronal calcium channels, Gem inhibit Cav2.3 channels in a CaVb-independent manner and through two synergistic mechanisms: a increase in voltage for activation and a decrease in the number of channels.

Human TRPM2 (hTRPM2) is a non-selective Ca 2þ permeable cation channel, expressed abundantly in brain, immune cells, and pancreatic b-cells. Under physiological conditions it plays a role in glucose-induced insulin secretion, and in the immune response. Besides, TRPM2 enhances the sensitivity of cells toward oxidative stress induced damage, e.g., facilitating neuronal cell death during brain ischemia/reperfusion. Nematostella vectensis TRPM2 (nvTRPM2) is an ancestor of hTRPM2. Both are activated by intracellular Ca 2þ and ADP-ribose (ADPR), and contain a C-terminal NUDT9-H domain, which shows high sequence homology with the mitochondrial ADPR-hydrolase enzyme NUDT9. We have recently shown that the NUDT9-H domain of hTRPM2 has lost its ADPRase activity due to mutations of the conserved ''Nudix box'' motif.Interestingly, in nvTRPM2 the Nudix-box motif is still canonical. Correspondingly, we found that purified nvTRPM2 protein, or its isolated NUDT9-H domain, shows robust ADPRase activity. We next addressed whether nvTRPM2 enzymatic activity plays any role in channel gating. Enzymatic activity of isolated nvNUDT9-H could be reduced or abolished by point mutations, as well as by Mg 2þ removal. On the other hand, in inside-out patch clamp experiments the kinetics of channel gating of full-length nvTRPM2 remained unaffected by these manipulations: none of them changed the apparent affinity of the currents towards ADPR, or the current relaxation time constant after sudden ADPR removal. We also investigated the effect of the non-hydrolyzable ADPR analogue AMPCPR on nvTRPM2 channel gating. Compared to ADPR, AMPCPR showed a lower apparent affinity, and caused shorter channel openings, suggesting that it fits less well into the binding pocket. These results together suggest that nvTRPM2 is a true channel-enzyme, but its enzymatic activity is not coupled to channel gating.

Intracellular calcium can modulate the kinetics of voltage-gated sodium (Nav) and other channels. Existing studies on Nav channels report effects on intrinsic kinetic properties, including inactivation. Here, we show evidence that calcium may also modulate the interaction between Nav channels and fibroblast growth factor-homologous factors (FHFs). Certain FHF subtypes bind to the C-terminus of Nav channels and result in an open-state block that competes with endogenous fast inactivation. This binding is fast (ms) at depolarizing voltages, but unbinding is very slow (100s of ms). Neurons that express these FHF subtypes have characteristically low firing rates. The calcium-dependent effect that we observe is mostly a change in the kinetics of recovery from the FHF-induced long-term inactivation. This calcium dependence may serve as an additional feedback mechanism for regulating neuronal firing rates.