Adiponectin receptor 1-mediated stimulation of Cav3.2 channels in trigeminal ganglion neurons induces nociceptive behaviors in mice

Abstract

Background Adipokines, including adiponectin, are implicated in nociceptive pain; however, the underlying cellular and molecular mechanisms remain unknown. Methods Using electrophysiological recording, immunostaining, molecular biological approaches and animal behaviour tests, we elucidated a pivotal role of adiponectin in regulating membrane excitability and pain sensitivity by manipulating Cav3.2 channels in trigeminal ganglion (TG) neurons. Results Adiponectin enhanced T-type Ca2+ channel currents (IT) in TG neurons through the activation of adiponectin receptor 1 (adipoR1) but independently of heterotrimeric G protein-mediated signaling. Coimmunoprecipitation revealed a physical association between AdipoR1 and casein kinase II alpha-subunits (CK2α) in the TG, and inhibiting CK2 activity by chemical inhibitor or siRNA targeting CK2α prevented the adiponectin-induced IT response. Adiponectin significantly activated protein kinase C (PKC), and this effect was abrogated by CK2α knockdown. Adiponectin increased the membrane abundance of PKC beta1 (PKCβ1). Blocking PKCβ1 pharmacologically or genetically abrogated the adiponectin-induced IT increase. In heterologous expression systems, activation of adipoR1 induced a selective enhancement of Cav3.2 channel currents, dependent on PKCβ1 signaling. Functionally, adiponectin increased TG neuronal excitability and induced mechanical pain hypersensitivity, both attenuated by T-type channel blockade. In a trigeminal neuralgia model induced by chronic constriction injury of infraorbital nerve, blockade of adipoR1 signaling suppressed mechanical allodynia, which was prevented by silencing Cav3.2. Conclusion Our study elucidates a novel signaling cascade wherein adiponectin stimulates TG Cav3.2 channels via adipoR1 coupled to a novel CK2α-dependent PKCβ1. This process induces neuronal hyperexcitability and pain hypersensitivity. Insight into adipoR-Cav3.2 signaling in sensory neurons provides attractive targets for pain treatment.

Melanopsin activates divergent phototransduction pathways in intrinsically photosensitive retinal ganglion cell subtypes

“…T-type VGCC mRNA expression has been reported in M2 ipRGCs ( Figure 9—figure supplement 1A ; Tran et al, 2019 ), and we detect T-type VGCC immunolabeling in ON-stratifying ipRGCs in retinal sections ( Figure 9—figure supplement 1B ). To test this functionally, we performed voltage clamp recordings of M2 cells stepping membrane voltage from –70 mV to more depolarized voltages (see 'Materials and methods' for detailed protocol) and compared current amplitudes in control solution and after application of the selective T-type VGCC antagonist TTA-P2 (10 µM) ( Figure 9A , Figure 9—figure supplement 2 ; Wu et al, 2018 ; Randall and Tsien, 1997 ; Timic Stamenic et al, 2019 ; Zhang et al, 2023 ; Dreyfus et al, 2010 ; Choe et al, 2011 ). We found that TTA-P2 reduced M2 current amplitudes in this protocol, and that TTA-P2-sensitive currents showed I–V relationships consistent with T-type VGCC channels ( Wu et al, 2018 ; Perez-Reyes, 2003 ; Randall and Tsien, 1997 ; Monteil et al, 2000 ; Fox et al, 1987 ; Dreyfus et al, 2010 ; Choe et al, 2011 ).…”

“…Before recording calcium currents, cells were voltage clamped at –70 mV and depolarized to –40 mV to block any transient channels. Cells were then stepped from –40 mV to voltages from –60 mV to +30 mV in 10 mV intervals ( Randall and Tsien, 1997 ; Dhein et al, 2004 ; Cai et al, 2019 ; Zhang et al, 2023 ).…”

“…M1 ipRGCs project to non-image-forming brain regions to influence subconscious non-image-forming functions such as circadian photoentrainment, the pupillary light reflex, learning, and mood ( Hattar et al, 2003 ; Lucas et al, 2003 ; Mrosovsky and Hattar, 2003 ; Panda et al, 2003 ; Altimus et al, 2008 ; Lupi et al, 2008 ; Göz et al, 2008 ; Gooley et al, 2012 ; LeGates et al, 2012 ; Fernandez et al, 2018 ; Rupp et al, 2019 ; Sondereker et al, 2020 ; Aranda and Schmidt, 2021 ). M2-M6 ipRGCs primarily innervate brain areas involved in conscious visual perception and are necessary for proper contrast sensitivity ( Ecker et al, 2010 ; Estevez et al, 2012 ; Zhang et al, 2023 ; Schmidt et al, 2014 ; Stabio et al, 2018 ; Quattrochi et al, 2019 ; Aranda and Schmidt, 2021 ). Despite diverse behavioral functions, one common feature across ipRGC subtypes is that melanopsin (Opn4) phototransduction is required for intrinsic light sensitivity.…”

“…Indeed, melanopsin null mutant animals show deficits in both image-forming and non-image-forming behaviors ( Panda et al, 2002 ; Ruby et al, 2002 ; Lucas et al, 2003 ; Schmidt et al, 2014 ), highlighting the important role melanopsin plays across diverse cell types and behaviors. Though all ipRGC subtypes require melanopsin phototransduction for intrinsic photosensitivity, the melanopsin-mediated response of each subtype differs in size, sensitivity, and kinetics and will differentially shape the visual information relayed to the brain ( Graham et al, 2008 ; Schmidt and Kofuji, 2009 ; Ecker et al, 2010 ; Perez-Leighton et al, 2011 ; Estevez et al, 2012 ; Zhang et al, 2023 ; Jiang et al, 2018 ; Sonoda et al, 2018 ; Stabio et al, 2018 ; Quattrochi et al, 2019 ). Yet, the mechanistic underpinnings of these key differences and how they impact visual signals relayed by ipRGC subtypes to drive diverse downstream behaviors are not well understood.…”

Interleukin-22 receptor 1-mediated stimulation of T-type Ca2+ channels enhances sensory neuronal excitability through the tyrosine-protein kinase Lyn-dependent PKA pathway

“…Immunoblot analysis was conducted as previously described [ 19 , 24 – 26 ]. Briefly, trigeminal ganglia were removed, dissected, and homogenized in radioimmunoprecipitation assay buffer (RIPA) buffer in the presence of proteinase inhibitor cocktail.…”