Abstract
Leptin regulates hypothalamic POMC+ (pro-opiomelanocortin) neurons by inducing TRPC (Transient Receptor Potential Cation) channel-mediate membrane depolarization. The role of TRPC channels in POMC neuron excitability is clearly established; however, it remains unknown whether their activity alone is sufficient to trigger excitability. Here we show that the right-shift voltage induced by the leptin-induced TRPC channel-mediated depolarization of the resting membrane potential brings T-type channels into the active window current range, resulting in an increase of the steady state T-type calcium current from 40 to 70% resulting in increased intrinsic excitability of POMC neurons. We assessed the role and timing of T-type channels on excitability and leptin-induced depolarization in vitro in cultured mouse POMC neurons. The involvement of TRPC channels in the leptin-induced excitability of POMC neurons was corroborated by using the TRPC channel inhibitor 2APB, which precluded the effect of leptin. We demonstrate T-type currents are indispensable for both processes, as treatment with NNC-55-0396 prevented the membrane depolarization and rheobase changes induced by leptin. Furthermore, co-immunoprecipitation experiments suggest that TRPC1/5 channels and CaV3.1 and CaV3.2 channels co-exist in complex. The functional relevance of this complex was corroborated using intracellular Ca2+ chelators; intracellular BAPTA (but not EGTA) application was sufficient to preclude POMC neuron excitability. However, leptin-induced depolarization still occurred in the presence of either BAPTA or EGTA suggesting that the calcium entry necessary to self-activate the TRPC1/5 complex is not blocked by the presence of BAPTA in hypothalamic neurons. Our study establishes T-type channels as integral part of the signaling cascade induced by leptin, modulating POMC neuron excitability. Leptin activation of TRPC channels existing in a macromolecular complex with T-type channels recruits the latter by locally induced membrane depolarization, further depolarizing POMC neurons, triggering action potentials and excitability.
Highlights
Leptin regulates energy homeostasis and serves as a satiety afferent signal in the homeostatic control of adipose tissue mass (Schwartz et al, 2000; Harvey and Ashford, 2003), reducing food intake, increasing energy expenditure and regulating the reward value of nutrient (Ahima and Flier, 2000; Domingos et al, 2011; Williams and Elmquist, 2012)
Immunocytochemistry analysis (ICC) showed that POMC and neuropeptide Y (NPY)/AGRP neurons were present in the culture, with POMC+ neurons (POMC) being the majority (85 vs. 15%, P < 0.05, Z-test) (Figures 1A,B); note that Figure 1A figure does not display an average distribution of POMC+ and AGRP+ positive neurons, as it was difficult to find such ratios within the same area during confocal image acquisition
We corroborated that our primary POMC+ hypothalamic neurons responded to leptin as expected according to the bibliography
Summary
Leptin regulates energy homeostasis and serves as a satiety afferent signal in the homeostatic control of adipose tissue mass (Schwartz et al, 2000; Harvey and Ashford, 2003), reducing food intake, increasing energy expenditure and regulating the reward value of nutrient (Ahima and Flier, 2000; Domingos et al, 2011; Williams and Elmquist, 2012). Anorexigenic POMC positive neurons are depolarized by leptin (Cowley et al, 2001). This depolarization is mediated via a Jak2-PI3 kinase-PLCγ pathway that activates TRPC channel activity (Qiu et al, 2010). A subset of POMC neurons in the arcuate nucleus responsive to serotonin via the 5HT2C receptor are activated via TRPC channels, suggesting TRPC channels are a common signaling mechanism mediating anorexigenic signaling in the hypothalamus (Sohn et al, 2011). TRPC5 channels are the molecular mediators of the acute leptin and serotonin effect in POMC neurons (Gao et al, 2017)
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