We made the surprise finding that loss of TMEM16C function led to the elimination of the majority of POA neurons that increase their firing rate with rising temperature of the preoptic area of the anterior hypothalamus (POA) ? warm-sensitive neurons that are on top of a command chain for thermoregulation. To test for the involvement of TMEM16C in thermoregulation and febrile seizure, we examined TMEM16C conditional knockout (cKO) mice and found that mutant pups with TMEM16C removed from their central neurons cannot maintain their body temperature and are prone to develop hyperthermia-induced seizure. To look for molecular markers of temperature sensitive POA neurons, we conducted single-cell RNAseq of 68 POA neurons following recording from these neurons in brain slices subjected to temperature changes, and validated a molecular and genetic marker for temperature sensitive POA neurons. By generating cKO mice with TMEM16C removed from temperature sensitive POA neurons, we aim to test the function of TMEM16C in the specification of warm-sensitive neurons, thermoregulation, and hyperthermia-induced seizure. Given the GWAS association of TMEM16C with febrile seizure and our finding that rodent pups without neuronal TMEM16C are prone to exhibit hyperthermia-induced seizures, TMEM16C cKO mice provide animal models for febrile seizure, which affects 2-8% of young children. Because complex febrile seizures are associated with hippocampal sclerosis as the epileptogenic pathology, we will test the hypothesis that TMEM16C regulates hippocampal neuronal excitability by recording action potentials and synaptic potentials in hippocampal neurons from mice with TMEM16C removed either via nestin-Cre that is expressed in all neurons or via Drd3-Cre that targets ~50% of hippocampal pyramidal neurons. We will also record from temperature sensitive POA neurons in TMEM16C cKO mice and sibling controls to determine how TMEM16C modulates temperature sensitive POA neuronal activity. We will further test the hypothesis that removing TMEM16C from central neurons causes alteration of sodium-activated potassium current in these neurons. Finally, we will adopt the recently developed split GFP approach involving the use of mass spectrometry to identify proteins that are associated with TMEM16C in specific neuronal types.
Having established that calcium-activated chloride channels are formed by TMEM16A or TMEM16B of the mammalian TMEM16 family with ten members, our curiosity about the function of their close relative TMEM16C led to the surprise finding that TMEM16C acts as an auxiliary subunit of the Slack (Kcnt1) sodium- activated potassium channel in nociceptive sensory neurons to modulate pain sensitivity, as well as a collaboration with Danish epidemiologists who identified a TMEM16C polymorphism in their GWAS study as a risk factor for febrile seizure. Having found that TMEM16C conditional knockout (cKO) mice provide a model for febrile seizure as manifested by an increased likelihood to exhibit hyperthermia-induced seizure, we proposed to test for the TMEM16C involvement in thermoregulation and regulation of central neuronal excitability.
