Discussion
It has been established that T-channels can contribute to the hyperexcitability of sensory neurons manifested by hyperalgesia and allodynia, two frequent symptoms of chronic neuropathic pain. Several studies have validated that plasticity of T-channels is implicated in hyperalgesia and allodynia in animal models of PDN. Taken together, these studies identify an important pronociceptive role of CaV3.2 T-channels in neuropathic pain in animal models of type 1 and type 2 diabetes.
However, molecular mechanisms for the alteration of CaV3.2 channels in DRG cells from diabetic animals have not previously been described. Indeed, herein, we provide evidence for the first time that targeting glycosylation states of T-channels may be a promising new treatment for painful PDN. This conclusion is based on several observations from our study. First, we show that macroscopic current activation and inactivation kinetics as well as current density are drastically reduced when recombinant human CaV3.2 channels expressed in HEK-293 cells reared in hyperglycemic cell culture medium are exposed to NEU and PNG. Second, the effects of NEU are more prominent in DRG cells from diabetic ob/ob mice than in healthy WT mice. Third, NEU in vivo completely reversed thermal and mechanical hyperalgesia in diabetic ob/ob mice, whereas it was completely ineffective in age-matched healthy WT mice. The fact that NEU modified DRG T-current kinetics from healthy WT mice but to a lesser extent than in ob/ob mice suggests that the level of T-channel glycosylation is an important physiological mechanism that fine-tunes the activity of these channels in pain pathways. However, it appears that this process is maladaptive and leads to cellular hyperexcitability and, consequently, hyperalgesia in diseases like PDN. Hence, glycosylation of CaV3.2 channels is an important mechanism of sensitization of peripheral nociceptors that could be exploited for novel pain therapies.
Our molecular studies identify conserved extracellular asparagine residues, most notably N192 and N1466, as important regulators of CaV3.2 current kinetics and channel membrane expression, respectively. This is supported by our patch-clamp recordings that demonstrated slower current kinetics in N192Q CaV3.2 mutant with apparently normal membrane expression. In contrast, we could not consistently record T-currents in HEK-293 cells transfected with N1466Q CaV3.2 mutant, and our imaging studies with the EGFP-tagged mutant showed minimal membrane expression. Thus, different glycosylation sites in CaV3.2 channels may have distinct functional roles. Surprisingly, we could not record T-currents from N271Q Cav3.2 channels despite apparently normal membrane expression. It remains possible that N271Q channels trafficked to the membrane are nonfunctional. During the review of our study, another in vitro study using recombinant human CaV3.2 channels also examined the effect of glycosylation on T-current kinetics and surface membrane expression. Surprisingly, they found that treatments with PNG but not NEU decreased CaV3.2 current density and slowed kinetics of channel inactivation. Furthermore, their work suggests that asparagine N192 serves as a regulator of channel membrane expression and asparagine N1466 as a regulator of channel kinetics. It is possible that specific conditions of enzymatic deglycosylation or different levels of glucose in cell culture could have contributed to the different findings between the studies. However, regardless of observed differences, our study directly demonstrates that CaV3.2 -channels are indeed glycosylated within domain I of the channel protein and for the first time reveals the prominent effects of NEU on native T-currents in DRG cells and on pain perception in vivo using an animal model of PDN.
Several other in vitro studies have reported that glycosylation may modulate properties of other voltage-gated ion channels. For example, in embryonic DRG neurons, NEU affected steady-state inactivation of voltage-gated sodium channels. While future biophysical studies may reveal fine details of the effects of glycosylation upon CaV3.2 current kinetics, it is reasonable to propose that increased current density and increased kinetics of CaV3.2 current activation alone may contribute to the hyperexcitable state of DRG cells under hyperglycemic conditions. Similar to the results of our study, the findings of Tyrrell et al. did not show any effects of NEU on voltage-gated sodium channels in small DRG cells from adult animals. Future extensive electrophysiological studies could be expanded to involve examination of other voltage-gated channels that are crucial for the control of cellular excitability of DRG cells from diabetic animals that might also be modulated by glycosylation.
Previous in situ hybridization studies and electrophysiological studies using KO mice have established that the CaV3.2 is the most prevalent isoform of the Cav3.0 family in small DRG cells. Thus, we have focused our investigation on the effects of glycosylation on this particular isoform and on the peripheral sensitization of pain responses in PDN. Our molecular studies have found that extracellular asparagine residues N192 and N1466 are likely putative substrates for glycosylation that alter T-channel membrane expression and current kinetics. Since these asparagine residues are conserved across all T-channel isoforms, it is likely that glycosylation may similarly modulate the other two T-channel isoforms, namely, CaV3.1 and CaV3.3. Interestingly, all three isoforms of T-type channels are expressed in dorsal horn neurons of spinal cord, and recent studies have shown that they all may support spinal nociceptive processing in different animal models of neuropathic pain. Thus, simultaneous glycosylation of multiple T-channel isoforms in spinal dorsal horn neurons may contribute to their hyperexcitability, which in turn may influence central sensitization of pain responses that is implicated in many pain disorders.
Overall, the results presented here fundamentally advance our understanding of the mechanisms of glycosylation underlying the posttranslational modification of CaV3.2 T-channels that has an important function in supporting peripheral nociceptive signaling. Our results strongly suggest that the manipulation of glycosylation states of peripheral nociceptors could be useful for the development of novel therapies for the treatment of painful PDN. This method may have an advantage over direct blockers of T-channels to suppress pain because NEU and related agents will correct the pathology of diabetes at its source rather than ameliorating the problem through separate pathways that also may be a source of unintended side effects. Our goal is to provide novel therapeutic modalities that would not only alleviate neuropathic pain in patients with diabetes but, even more importantly, also halt its progression without causing dangerous systemic side effects or creating the potential for drug abuse.