Calpain cleaves the carboxyl terminus of TRPV1 and modulates receptor tachyphylaxis
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Abstract
Transient receptor potential vanilloid-1 (TRPV1) plays a critical role in noxious heat sensation and pain hypersensitivity development in chronic pain. Sustained or repeated exposure to capsaicin, a classic TRPV1 agonist, induces TRPV1 desensitization. This partially accounts for the analgesic effect of capsaicin. However, the regulatory mechanisms of TRPV1 desensitization remain poorly understood. In this study, we found that capsaicin acts on TRPV1 to induce the activation of calpain, a Ca 2+ -sensitive cysteine protease, in a manner unrelated to cellular injury. Calpain cleaves rTRPV1 at the carboxyl terminus of G819/S820. Lack of the distal carboxyl terminus leads to reduced TRPV1 localization on the plasma membrane, potentially due to increased receptor internalization and impaired subunit assembly. This finding was corroborated by whole-cell patch clamp recordings. Additionally, the Δ820-838 mutant of rTRPV1 shows resistance to tachyphylaxis as induced by repetitive capsaicin stimulation. This study reveals a pivotal role for calpain in TRPV1 desensitization where its activation constrains TRPV1 channel function while simultaneously increasing its resistance to tachyphylaxis, thereby acting to maintain TRPV1 activity within an appropriate range.
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In the current work, we demonstrated that TRPV1-Ca2+-calpain pathway activated by a regular dose (1 μM) of capsaicin is not associated with cell toxic injury
What do you mean by "regular dose"? It might be helpful to think about this in terms relative to the Kd of capsaicin for TRPV1.
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Consequently, TRPV1 Δ820 displayed a similar activation pattern as WT TRPV1 when subjected to voltage stimulation
Do you have a negative control for this experiment, where TRPV1 is absent from the cells?
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From these findings, we could conjecture that calpain-mediated cleavage of TRPV1 may promote receptor internalization.
Can you be more direct here and use this assay to show calpain is cleaving TRPV1 and that this leads to receptor internalization? For instance, addition of capsaicin should lead to activation of calpain that should cleave TRPV1, according to your theory. If so, after activation, can you detect the cleaved version of TRPV1 being internalized?
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This lack of impact might be caused by the feature of calpain where it recognizes substrates not only by the primary sequence of their amino acids, but also by their overall three-dimensional structure
Given that mutation of residues that were predicted to be critical for cleavage had no effect, it would be important to confirm that this site is cleaved in vivo. This could rule out that the cleavage you observe in vitro is not an artifact due to incubation with high levels of enzyme or overepxression of substrates.
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For this, N-terminal GST-tag or C-terminal GFP-tag TRPV1 was transiently transfected into human embryonic kidney (HEK) 293 cells.
This is a very intriguing idea linking TRPV1-mediated calpain activation to downregulation of TRPV1! While your engineered HEK and CHO cell systems work well, can you perform this assay in more biologically relevant cells, such as DRGs, or cells more closely related to neurons, like keratinocytes, and examine endogenous proteins?
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