Calmodulin (CaM) regulates gating of several types of ion channels but has not been implicated in channel assembly or trafficking. For the SK4/IK1 K+ channel, CaM bound to the proximal C terminus ("Ct1 " domain) acts as the Ca2+ sensor. We now show that CaM interacting with the C terminus of SK4 also controls channel assembly and surface expression. In transfected cells, removing free CaM by overexpressing the CaM-binding domain, Ct1, redistributed full-length SK4 protein from the plasma membrane to the cytoplasm and decreased whole-cell currents. Making more CaM protein available by overexpressing the CaM gene abrogated the dominant-negative effect of Ct1 and restored both surface expression of SK4 protein and whole-cell currents. The distal C-terminal domain ("Ct2") also plays a role in assembly, but is not CaM-dependent. Co-immunoprecipitation experiments demonstrated that multimerization of SK4 subunits was enhanced by CaM and inhibited by removal of CaM, indicating that CaM regulates trafficking of SK4 by affecting the assembly of channels. Our results support a model in which CaM-dependent association of SK4 monomers at their Ct1 domains regulates channel assembly and surface expression. This appears to represent a novel mechanism for controlling ion channels, and consequently, the cellular functions that depend on them.
The N-type voltage-gated calcium channel (CaV2.2) is a clinically endorsed target in chronic pain treatments. As directly targeting the channel can lead to multiple adverse side effects, targeting modulators of CaV2.2 may prove better. We previously identified ST1-104, a short peptide from the collapsin response mediator protein 2 (CRMP2), which disrupted the CaV2.2-CRMP2 interaction and suppressed a model of HIV-related neuropathy induced by anti-retroviral therapy but not traumatic neuropathy. Here, we report ST2-104 -a peptide wherein the cell-penetrating TAT motif has been supplanted with a homopolyarginine motif, which dose-dependently inhibits the CaV2.2-CRMP2 interaction and inhibits depolarization-evoked Ca(2+) influx in sensory neurons. Ca(2+) influx via activation of vanilloid receptors is not affected by either peptide. Systemic administration of ST2-104 does not affect thermal or tactile nociceptive behavioral changes. Importantly, ST2-104 transiently reduces persistent mechanical hypersensitivity induced by systemic administration of the anti-retroviral drug 2',3'-dideoxycytidine (ddC) and following tibial nerve injury (TNI). Possible mechanistic explanations for the broader efficacy of ST2-104 are discussed.
Aberrant ion channel function has been heralded as a main underlying mechanism driving epilepsy and its symptoms. However, it has become increasingly clear that treatment strategies targeting voltage-gated sodium or calcium channels merely mask the symptoms of epilepsy without providing disease-modifying benefits. Ion channel function is likely only one important cog in a highly complex machine. Gross morphological changes, such as reactive sprouting and outgrowth, may also play a role in epileptogenesis. Mechanisms responsible for these changes are not well-understood. Here we investigate the potential involvement of the neurite outgrowth-promoting molecule collapsin response mediator protein 2 (CRMP2). CRMP2 activity, in this respect, is regulated by phosphorylation state, where phosphorylation by a variety of kinases, including glycogen synthase kinase 3 β (GSK3β) renders it inactive. Phosphorylation (inactivation) of CRMP2 was decreased at two distinct phases following traumatic brain injury (TBI). While reduced CRMP2 phosphorylation during the early phase was attributed to the inactivation of GSK3β, the sustained decrease in CRMP2 phosphorylation in the late phase appeared to be independent of GSK3β activity. Instead, the reduction in GSK3β-phosphorylated CRMP2 was attributed to a loss of priming by cyclin-dependent kinase 5 (CDK5), which allows for subsequent phosphorylation by GSK3β. Based on the observation that the proportion of active CRMP2 is increased for up to 4 weeks following TBI, it was hypothesized that it may drive neurite outgrowth, and therefore, circuit reorganization during this time. Therefore, a novel small-molecule tool was used to target CRMP2 in an attempt to determine its importance in mossy fiber sprouting following TBI. In this report, we demonstrate novel differential regulation of CRMP2 phosphorylation by GSK3β and CDK5 following TBI.
Epileptogenesis following traumatic brain injury (TBI) is likely due to a combination of increased excitability, disinhibition, and increased excitatory connectivity via aberrant axon sprouting. Targeting these pathways could be beneficial in the prevention and treatment of posttraumatic epilepsy. Here, we tested this possibility using the novel anticonvulsant (R)-N-benzyl 2-acetamido-3-methoxypropionamide ((R)-lacosamide [LCM]), which acts on both voltage-gated sodium channels and collapsin response mediator protein 2 (CRMP2), an axonal growth/guidance protein. LCM inhibited CRMP2-mediated neurite outgrowth, an effect phenocopied by CRMP2 knockdown. Mutation of LCM-binding sites in CRMP2 reduced the neurite inhibitory effect of LCM by ∼8-fold. LCM also reduced CRMP2-mediated tubulin polymerization. Thus, LCM selectively impairs CRMP2-mediated microtubule polymerization, which underlies its neurite outgrowth and branching. To determine whether LCM inhibits axon sprouting in vivo, LCM was injected into rats subjected to partial cortical isolation, an animal model of posttraumatic epileptogenesis that exhibits axon sprouting in cortical pyramidal neurons. Two weeks following injury, excitatory synaptic connectivity of cortical layer V pyramidal neurons was mapped using patch clamp recordings and laser scanning photostimulation of caged glutamate. In comparison with injured control animals, there was a significant decrease in the map size of excitatory synaptic connectivity in LCM-treated rats, suggesting that LCM treatment prevented enhanced excitatory synaptic connectivity due to posttraumatic axon sprouting. These findings suggest, for the first time, that LCM's mode of action involves interactions with CRMP2 to inhibit posttraumatic axon sprouting.
The ubiquity of protein-protein interactions in biological signaling offers ample opportunities for therapeutic intervention. We previously identified a peptide, designated CBD3, that suppressed inflammatory and neuropathic behavioral hypersensitivity in rodents by inhibiting the ability of collapsin response mediator protein 2 (CRMP-2) to bind to N-type voltage-activated calcium channels (CaV2.2) [Brittain et al. Nature Medicine 17:822-829 (2011)].