KLHL1 Controls CaV3.2 Expression in DRG Neurons and Mechanical Sensitivity to Pain

Abstract

Dorsal root ganglion (DRG) neurons process pain signaling through specialized nociceptors located in their peripheral endings. It has long been established low voltage-activated (LVA) Ca V 3.2 calcium channels control neuronal excitability during sensory perception in these neurons. Silencing Ca V 3.2 activity with antisense RNA or genetic ablation results in anti-nociceptive, anti-hyperalgesic and anti-allodynic effects. Ca V 3.2 channels are regulated by many proteins (Weiss and Zamponi, 2017), including KLHL1, a neuronal actin-binding protein that stabilizes channel activity by recycling it back to the plasma membrane through the recycling endosome. We explored whether manipulation of KLHL1 levels and thereby function as a Ca V 3.2 modifier can modulate DRG excitability and mechanical pain transmission or sensitivity to pain. We first assessed the mechanical sensitivity threshold and DRG properties in the KLHL1 KO mouse model. KO DRG neurons exhibited smaller T-type current density compared to WT without significant changes in voltage dependence, as expected in the absence of its modulator. Western blot analysis confirmed Ca V 3.2 but not Ca V 3.1, Ca V 3.3, Ca V 2.1, or Ca V 2.2 protein levels were significantly decreased; and reduced neuron excitability and decreased pain sensitivity were also found in the KLHL1 KO model. Analogously, transient down-regulation of KLHL1 levels in WT mice with viral delivery of anti-KLHL1 shRNA also resulted in decreased pain sensitivity. These two experimental approaches confirm KLHL1 as a physiological modulator of excitability and pain sensitivity, providing a novel target to control peripheral pain.

Targeting the T-type calcium channel Cav3.2 in GABAergic arcuate nucleus neurons to treat obesity

“…The activity dynamics of GABAergic neurons in different hypothalamic regions, including ARH [ 28 , 29 ], lateral hypothalamus (LH) [ 30 , 31 ], tuberal nucleus (TN) [ 32 , 33 ], and zona incerta (ZI) [ 34 , 35 ], plays a vital role in the regulation of feeding behavior, body weight, and energy expenditure. It has been demonstrated that neural excitability is regulated by subtypes of neuronal voltage-activated Ca 2+ channels, including CAV3.2 [ 36 , 37 ]. CAV3.2 may serve as a critical intrinsic modulator for the neural excitability of hypothalamic GABAergic neurons to control energy homeostasis.…”

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

“…KLHL1 KO mice display similar alterations but they also exhibit compensatory expression to sustain calcium current homeostasis in hippocampal neurons, yet they still display synaptic alterations ( Perissinotti et al, 2015 ). Moreover, DRG neurons from KLHL1 KO mice exhibit and uncompensated reduction of Ca V 3.2 expression, resulting in reduced neuron excitability and decreased sensitivity to pain ( Martínez-Hernández et al, 2020 ). Thus, deletion of KLHL1 results in differential alteration of calcium currents and neuronal excitability depending on their role in specific nervous system areas.…”

Targeting intrinsically disordered regions facilitates discovery of calcium channels 3.2 inhibitory peptides for adeno-associated virus–mediated peripheral analgesia

“… 13 , 46 , 75 Although our studies have initially focused on testing whether targeting IDRs can facilitate discovery of T-type/Ca V 3.2 inhibitory peptides for AAV-mediated analgesia, how the identified Ca V 3.2iPA functioning remains unknown. The potential signaling pathways that the Ca V 3.2iPAs affected could be many because Ca V 3.2 intracellular segments serve as essential interfaces for many regulatory signaling molecules, including nuclear-localized deubiquitinating enzyme (USP5), 29 calcium/calmodulin–dependent protein kinase II, 35 cyclin-dependent kinase 5, 32 G-proteins, 64 calcineurin, 34 calmodulin, 15 syntaxin/SNAP25, 83 Kelch-like protein 1, 50 and Stac1. 74 In addition, Ca V 3.2 can form protein complexes with members of the K + channel family, such as K V 4, K Ca3.1 , and K Ca1.1 (BK), 2 , 70 and K + /Na + hyperpolarization–activated cyclic nucleotide-gated channel 1, 22 as well as lipids.…”