N-Glycosylation of TRPM8 Ion Channels Modulates Temperature Sensitivity of Cold Thermoreceptor Neurons

Pertusa, Marı́a; Madrid, Rodolfo; Morenilla‐Palao, Cruz; Belmonte, Carlos; Viana, Félix

doi:10.1074/jbc.m111.312645

Cited by 67 publications

(87 citation statements)

References 54 publications

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“…Beyond their role in protein biogenesis and/or stability (37), N-linked glycans promote interactions with cell adhesion proteins and signaling molecules (40,89) as it is widely understood for members of the integrin family (90). Glycans could also contribute to channel modulation by altering the surface potential sensed by the gating machinery (92) and/or by modifying conformational changes regulating cooperative subunit interactions during channel activation (93). The Asn-812 site is a choice candidate for such a mechanism.…”

Section: Discussionmentioning

confidence: 99%

Identification of Glycosylation Sites Essential for Surface Expression of the CaVα2δ1 Subunit and Modulation of the Cardiac CaV1.2 Channel Activity

Tétreault¹,

Bourdin²,

Briot³

et al. 2016

Journal of Biological Chemistry

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Alteration in the L-type current density is one aspect of the electrical remodeling observed in patients suffering from cardiac arrhythmias. Changes in channel function could result from variations in the protein biogenesis, stability, post-translational modification, and/or trafficking in any of the regulatory subunits forming cardiac L-type Ca 2؉ channel complexes. Ca V ␣2␦1 is potentially the most heavily N-glycosylated subunit in the cardiac L-type Ca V 1.2 channel complex. Here, we show that enzymatic removal of N-glycans produced a 50-kDa shift in the mobility of cardiac and recombinant Ca V ␣2␦1 proteins. This change was also observed upon simultaneous mutation of the 16 Asn sites. Nonetheless, the mutation of only 6/16 sites was sufficient to significantly 1) reduce the steady-state cell surface fluorescence of Ca V ␣2␦1 as characterized by two-color flow cytometry assays and confocal imaging; 2) decrease protein stability estimated from cycloheximide chase assays; and 3) prevent the Ca V ␣2␦1-mediated increase in the peak current density and voltage-dependent gating of Ca V 1.2. Reversing the N348Q and N812Q mutations in the non-operational sextuplet Asn mutant protein partially restored Ca V ␣2␦1 function. Single mutation N663Q and double mutations N348Q/N468Q, N348Q/N812Q, and N468Q/N812Q decreased protein stability/synthesis and nearly abolished steady-state cell surface density of Ca V ␣2␦1 as well as the Ca V ␣2␦1-induced up-regulation of L-type currents. These results demonstrate that Asn-663 and to a lesser extent Asn-348, Asn-468, and Asn-812 contribute to protein stability/synthesis of Ca V ␣2␦1, and furthermore that N-glycosylation of Ca V ␣2␦1 is essential to produce functional L-type Ca 2؉ channels.The regulation of Ca 2ϩ influx in cardiac cells is critical to the generation of the force necessary for the myocardium to meet the physiological needs of the body (1). In resting cells, intracellular free ionized Ca 2ϩ is maintained at a low concentration (high nanomolar range) by the concerted action of mechanisms that prevent Ca 2ϩ entry, promote its extrusion (mostly via the Na ϩ /Ca 2ϩ exchanger), and ensure its storage in the sarcoplasmic reticulum (2). Ca 2ϩ entry is mediated mainly by the cardiac L-type Ca 2ϩ channel, which is central to the initiation of excitation-contraction coupling via Ca 2ϩ -induced Ca 2ϩ release from the sarcoplasmic reticulum. Regulation of the L-type Ca 2ϩ current has profound physiological significance. Indeed, alterations in density or the activation/inactivation gating of L-type Ca 2ϩ channels have been implicated in a variety of cardiovascular diseases (3, 4), including cardiac arrhythmias such as atrial fibrillation (5-8), heart failure (9, 10), and ischemic heart disease (10). The molecular mechanisms underlying changes in the activity of the L-type Ca 2ϩ channel remain under study for most pathologies.The L-type Ca V 1.2 channel belongs to the molecular family of high voltage-activated Ca V channels. High voltage-activated Ca V 1.2 channels are hetero-oligo...

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Section: Discussionmentioning

confidence: 99%

Identification of Glycosylation Sites Essential for Surface Expression of the CaVα2δ1 Subunit and Modulation of the Cardiac CaV1.2 Channel Activity

Tétreault¹,

Bourdin²,

Briot³

et al. 2016

Journal of Biological Chemistry

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“…Several cellular processes modify temperature sensitivity and gating of TRPM8 in vitro. Post-translational N-glycosylation (Pertusa et al, 2012) and phosphatidylinositol 4,5-bisphosphate modulation (Rohacs et al, 2005) shift the voltage dependence of TRPM8, increasing the probability of opening at physiologic temperatures. Neurotrophic factors such as artemin and nerve growth factor also sensitize coldevoked behaviors in a TRPM8-dependent manner .…”

Section: Discussionmentioning

confidence: 99%

Novel TRPM8 Antagonist Attenuates Cold Hypersensitivity after Peripheral Nerve Injury in Rats

Patel

Gonçalves

Newman³

et al. 2014

J Pharmacol Exp Ther

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Abnormal cold sensitivity is a common feature of a range of neuropathies. In the murine somatosensory system, multiple aspects of cold sensitivity are dependent on TRPM8, both short term and in response to peripheral nerve injury. The specialized nature of cold-sensitive afferents and the restricted expression of TRPM8 render it an attractive target for the treatment of cold hypersensitivity. This current study examines the effect of a novel TRPM8 antagonist (M8-An) in naive and spinal nerveligated rats through behavioral and in vivo electrophysiological approaches. In vitro, M8-An inhibited icilin-evoked Ca 21 currents in HEK293 cells stably expressing human TRPM8 with an IC 50 of 10.9 nM. In vivo, systemic M8-An transiently decreased core body temperature. Deep dorsal horn recordings were made in vivo from neurons innervating the hind paw. M8-An inhibited neuronal responses to innocuous and noxious cooling of the receptive field in spinal nerve-ligated rats but not in naive rats. No effect on neuronal responses to mechanical and heat stimulation was observed. In addition, M8-An also attenuated behavioral responses to cold but not mechanical stimulation after nerve ligation without affecting the uninjured contralateral response. The data presented here support a contribution of TRPM8 to the pathophysiology of cold hypersensitivity in this model and highlight the potential of the pharmacological block of TRPM8 in alleviating the associated symptoms.

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“…Indeed, we showed previously that, in transfected HEK293 cells, functional TRPM8 activity from the ER can be detected by calcium imaging when cold and menthol stimuli are applied simultaneously (i.e. using a saturating stimulus) (32). Therefore, in the hypothetical case that TRPM8 mutant subunits are properly folded and the homotetramer is correctly assembled, we should observe a response to a combined cold and menthol stimulation even if the channel is mainly localized in the ER, suggesting that the deletion would be affecting primarily the normal trafficking of the channel.…”

Section: Distal N-terminal Region Encompassing Positions 40 -60 Ismentioning

confidence: 99%

“…TRPM8 is also N-glycosylated in this extracellular loop, specifically on the Asn residue at position 934, near the pore (26,30). This post-translational modification, also observed in native channels (31,32), plays a role in localizing TRPM8 channels on lipid rafts (31), and it is an important determinant of TRPM8 sensitivity to chemical and thermal stimuli (32). Most of TRPM8-binding sites for chemical agonists and antagonists have been mapped on transmembrane domains.…”

mentioning

confidence: 99%

Bidirectional Modulation of Thermal and Chemical Sensitivity of TRPM8 Channels by the Initial Region of the N-terminal Domain

Pertusa

González

Hardy

et al. 2014

Journal of Biological Chemistry

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Background:The functional role of the TRPM8 N terminus remains poorly understood. Results: Truncation of the first 40 residues increases TRPM8 responses to cold and menthol, whereas deletions within the 40 -60-residue region yield nonfunctional channels retained in the ER. Conclusion: Initial N terminus of TRPM8 is important for proper biogenesis and function. Significance: Variations within the N terminus can modulate thermal and chemical sensitivity of TRPM8.

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N-Glycosylation of TRPM8 Ion Channels Modulates Temperature Sensitivity of Cold Thermoreceptor Neurons

Cited by 67 publications

References 54 publications

Identification of Glycosylation Sites Essential for Surface Expression of the CaVα2δ1 Subunit and Modulation of the Cardiac CaV1.2 Channel Activity

Identification of Glycosylation Sites Essential for Surface Expression of the CaVα2δ1 Subunit and Modulation of the Cardiac CaV1.2 Channel Activity

Novel TRPM8 Antagonist Attenuates Cold Hypersensitivity after Peripheral Nerve Injury in Rats

Bidirectional Modulation of Thermal and Chemical Sensitivity of TRPM8 Channels by the Initial Region of the N-terminal Domain

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