TRPC1/5-CaV3 Complex Mediates Leptin-Induced Excitability in Hypothalamic Neurons

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.

Genetic Deletion of KLHL1 Leads to Hyperexcitability in Hypothalamic POMC Neurons and Lack of Electrical Responses to Leptin

“…Cultured hypothalamic neurons were studied from 8-10 days in vitro (DIV). Immunocytochemistry analysis from KLHL1-KO (KLHL1 –/– ) cultures yielded similar results to our previous analysis of WT neurons ( Perissinotti et al, 2021 ). From a total of 44 neurons, the distribution of POMC + vs .…”

“…Lastly, steady-state analysis shows that V 50 values for activation (SSA) and inactivation (SSI) were both discretely but significantly shifted ∼−2 mV in KLHL1-KO compared to WT neurons ( Figure 3E ; P < 0.05, t -test). SSA V 50 was −41.8 ± 0.7 mV in WT ( Perissinotti et al, 2021 ) compared to −44.3 ± 0.9 mV in KLHL1-KO ( P < 0.05, t -test); no change in the slope factor k was observed (5.2 ± 0.3 for WT and 5.1 ± 0.3 for KO). Comparably, SSI V 50 changed from −65.4 ± 1.0 mV in WT ( Perissinotti et al, 2021 ) to −67.5 ± 0.7 mV in KO ( P < 0.05, t -test), and the slope factor k changed from 7.1 ± 0.5 to 6.1 ± 0.2 ( P < 0.05, t -test).…”

“…We analyzed the response to leptin in a subset of WT pyramidal neurons using electrophysiology (other neuronal morphologies were not explored); from a total of 19 neurons, 79% responded positively to leptin (excitable), 5% were inhibited and 16% did not respond ( Figure 2E ), as we previously reported ( Perissinotti et al, 2021 ). Neuron cultures from KLHL1 –/– /EGFP-POMC +/– mice were used to verify the identity of POMC neurons ( Figure 2F ).…”

“…The hypothalamus plays an important regulatory role in feeding, TRPC1/5 and Ca V 3.1/2 (α 1 G /α 1 H ) channels are crucial components in the signal transduction pathway induced by leptin that leads to neuronal excitability ( Figure 1A ; Qiu et al, 2010 ; Perissinotti et al, 2021 ). We explored the expression levels of these channels in the KLHL1-KO hypothalamus, as this mice model displays compensatory changes in calcium channel expression and excitability in other brain areas.…”

“…T-type channels (Ca V 3.1 or Ca V 3.2) exist in a macromolecular complex with TRPC1/5 channels. TRPC1/5 channel activity is recruited by PLCγ, generating membrane depolarization, T-type channel-initiated action potentials and increased excitability ( Qiu et al, 2010 ; Perissinotti et al, 2021 ).…”