Faculty Bibliography

The use of N-type voltage-gated calcium channel (CaV2.2) blockers to treat pain is limited by many physiological side effects. Here we report that inflammatory and neuropathic hypersensitivity can be suppressed by inhibiting the binding of collapsin response mediator protein 2 (CRMP-2) to CaV2.2 and thereby reducing channel function. A peptide of CRMP-2 fused to the HIV transactivator of transcription (TAT) protein (TAT-CBD3) decreased neuropeptide release from sensory neurons and excitatory synaptic transmission in dorsal horn neurons, reduced meningeal blood flow, reduced nocifensive behavior induced by formalin injection or corneal capsaicin application and reversed neuropathic hypersensitivity produced by an antiretroviral drug. TAT-CBD3 was mildly anxiolytic without affecting memory retrieval, sensorimotor function or depression. At doses tenfold higher than that required to reduce hypersensitivity in vivo, TAT-CBD3 caused a transient episode of tail kinking and body contortion. By preventing CRMP-2-mediated enhancement of CaV2.2 function, TAT-CBD3 alleviated inflammatory and neuropathic hypersensitivity, an approach that may prove useful in managing chronic pain.

The novel anti-epileptic drug lacosamide targets two proteins - voltage-gated sodium channels and collapsin response mediator protein 2 (CRMP-2) - suggesting dual modes of action for lacosamide. We recently identified the neurite outgrowth and axonal guidance protein CRMP-2 as a novel partner and regulator of the presynaptic N-type voltage-gated Ca(2+) channel (CaV2.2) [Brittain et al., J. Biol. Chem. 284: 31375-31390 (2009)]. Here we examined the effects of lacosamide on voltage-gated Ba(2+) channels. Lacosamide did not affect Ba(2+) currents via N- and P/Q- channels in rat hippocampal neurons or L-type Ca(2+) channels in a mouse CNS neuronal cell line, respectively. N-type Ba(2+) currents, augmented by CRMP-2 expression, were also unaffected by acute or chronic lacosamide exposure. These results establish that the anti-epileptic mode of action of lacosamide does not involve these voltage-gated Ca(2+) channels.

The novel antiepileptic drug, (R)-N-benzyl 2-acetamido-3-methoxypropionamide ((R)-lacosamide, Vimpat(®) ((R)-1)), was recently approved in the US and Europe for adjuvant treatment of partial-onset seizures in adults. (R)-1 preferentially enhances slow inactivation of voltage-gated Na(+) currents, a pharmacological process relevant in the hyperexcitable neuron. We have advanced a strategy to identify lacosamide binding partners by attaching affinity bait (AB) and chemical reporter (CR) groups to (R)-1 to aid receptor detection and isolation. We showed that select lacosamide AB and AB&CR derivatives exhibited excellent activities similar to (R)-1 in the maximal electroshock seizure model in rodents. Here, we examined the effect of these lacosamide AB and AB&CR derivatives and compared them with (R)-1 on Na(+) channel function in CNS catecholaminergic (CAD) cells. Using whole-cell patch clamp electrophysiology, we demonstrated that the test compounds do not affect the Na(+) channel fast inactivation process, that they were far better modulators of slow inactivation than (R)-1, and that modulation of the slow inactivation process was stereospecific. The lacosamide AB agents that contained either an electrophilic isothiocyanate ((R)-5) or a photolabile azide ((R)-8) unit upon AB activation gave modest levels of permanent Na(+) channel slow inactivation, providing initial evidence that these compounds may have covalently reacted with their cognate receptor(s). Our findings support the further use of these agents to delineate the (R)-1-mediated Na(+) channel slow inactivation process.

The anti-epileptic drug (R)-lacosamide ((2R)-2-(acetylamino)-N-benzyl-3-methoxypropanamide (LCM)) modulates voltage-gated sodium channels (VGSCs) by preferentially interacting with slow inactivated sodium channels, but the observation that LCM binds to collapsin response mediator protein 2 (CRMP-2) suggests additional mechanisms of action for LCM. We postulated that CRMP-2 levels affects the actions of LCM on VGSCs. CRMP-2 labeling by LCM analogs was competitively displaced by excess LCM in rat brain lysates. Manipulation of CRMP-2 levels in the neuronal model system CAD cells affected slow inactivation of VGSCs without any effects on other voltage-dependent properties. In silico docking was performed to identify putative binding sites in CRMP-2 that may modulate the effects of LCM on VGSCs. These studies identified five cavities in CRMP-2 that can accommodate LCM. CRMP-2 alanine mutants of key residues within these cavities were functionally similar to wild-type CRMP-2 as assessed by similar levels of enhancement in dendritic complexity of cortical neurons. Next, we examined the effects of expression of wild-type and mutant CRMP-2 constructs on voltage-sensitive properties of VGSCs in CAD cells: 1) steady-state voltage-dependent activation and fast-inactivation properties were not affected by LCM, 2) CRMP-2 single alanine mutants reduced the LCM-mediated effects on the ability of endogenous Na(+) channels to transition to a slow inactivated state, and 3) a quintuplicate CRMP-2 alanine mutant further decreased this slow inactivated fraction. Collectively, these results identify key CRMP-2 residues that can coordinate LCM binding thus making it more effective on its primary clinical target.

Mutations of the neurofibromin gene (NF1) cause neurofibromatosis type 1 (NF1), a disease in which learning disabilities are common. Learning deficits also are observed in mice with a heterozygous mutation of Nf1 (Nf1(+/-)). Dysregulation of regulated neurotransmitter release has been observed in Nf1(+/-) mice. However, the role of presynaptic voltage-gated Ca(2+) channels mediating this release has not been investigated. We investigated whether Ca(2+) currents and transmitter release were affected by reduced neurofibromin in Nf1(+/-) mice. Hippocampal Ca(2+) current density was greater in neurons from Nf1(+/-) mice and a greater fraction of Ca(2+) currents was activated at less depolarized potentials. In addition, release of the excitatory neurotransmitter, glutamate, was increased in neuronal cortical cultures from Nf1(+/-) mice. Dendritic complexity and axonal length were also increased in neurons Nf1(+/-) mice compared to wild-type neurons, linking loss of neurofibromin to developmental changes in hippocampal axonal/cytoskeletal dynamics. Collectively, these results show that altered Ca(2+) channel density and transmitter release, along with increased axonal growth may account for the abnormal nervous system functioning in NF1.

Presynaptic N-type voltage-gated Ca(2+) channels (Cav2.2) form part of an extensive macromolecular complex in the presynaptic terminal. Regulation of Cav2.2 is achieved via protein-protein interactions within the terminal and can directly impact transmitter release which is dependent on Ca(2+) influx via these Cav2.2. We recently identified a novel Cav2.2 interacting partner-the collapsin response mediator protein (CRMP).1 CRMPs are a family of five proteins implicated in signal transduction of neurite outgrowth and axonal guidance. We showed that CRMP-2, a wellstudied member of this family, interacted with Cav2.2 via direct binding to cytoplasmic loops of Cav2.2. Depolarization enhanced the interaction. Further studies revealed that CRMP-2 facilitated an increase in Cav2.2 current density by inserting more Cav2.2 at the cell surface. As a consequence of CRMP-2-mediated increase in Ca(2+) influx, release of the excitatory neurotransmitter glutamate was also increased. CRMP-2 localized to synapses where, surprisingly, its overexpression increased synapse size. We hypothesize that the CRMP-2-calcium channel interaction represents a novel mechanism for modulation of Ca(2+) influx into nerve terminals and, hence, of synaptic strength. In this addendum, we further discuss the significance of this study and the possible implications to the field.

Collapsin response mediator proteins (CRMPs) mediate signal transduction of neurite outgrowth and axonal guidance during neuronal development. Voltage-gated Ca(2+) channels and interacting proteins are essential in neuronal signaling and synaptic transmission during this period. We recently identified the presynaptic N-type voltage-gated Ca(2+) channel (Cav2.2) as a CRMP-2-interacting partner. Here, we investigated the effects of a functional association of CRMP-2 with Cav2.2 in sensory neurons. Cav2.2 colocalized with CRMP-2 at immature synapses and growth cones, in mature synapses and in cell bodies of dorsal root ganglion (DRG) neurons. Co-immunoprecipitation experiments showed that CRMP-2 associates with Cav2.2 from DRG lysates. Overexpression of CRMP-2 fused to enhanced green fluorescent protein (EGFP) in DRG neurons, via nucleofection, resulted in a significant increase in Cav2.2 current density compared with cells expressing EGFP. CRMP-2 manipulation changed the surface levels of Cav2.2. Because CRMP-2 is localized to synaptophysin-positive puncta in dense DRG cultures, we tested whether this CRMP-2-mediated alteration of Ca(2+) currents culminated in changes in synaptic transmission. Following a brief high-K(+)-induced stimulation, these puncta became loaded with FM4-64 dye. In EGFP and neurons expressing CRMP-2-EGFP, similar densities of FM-loaded puncta were observed. Finally, CRMP-2 overexpression in DRG increased release of the immunoreactive neurotransmitter calcitonin gene-related peptide (iCGRP) by approximately 70%, whereas siRNA targeting CRMP-2 significantly reduced release of iCGRP by approximately 54% compared with control cultures. These findings support a novel role for CRMP-2 in the regulation of N-type Ca(2+) channels and in transmitter release.

Collapsin response mediator proteins (CRMPs) specify axon/dendrite fate and axonal growth of neurons through protein-protein interactions. Their functions in presynaptic biology remain unknown. Here, we identify the presynaptic N-type Ca(2+) channel (CaV2.2) as a CRMP-2-interacting protein. CRMP-2 binds directly to CaV2.2 in two regions: the channel domain I-II intracellular loop and the distal C terminus. Both proteins co-localize within presynaptic sites in hippocampal neurons. Overexpression in hippocampal neurons of a CRMP-2 protein fused to enhanced green fluorescent protein caused a significant increase in Ca(2+) channel current density, whereas lentivirus-mediated CRMP-2 knockdown abolished this effect. Interestingly, the increase in Ca(2+) current density was not due to a change in channel gating. Rather, cell surface biotinylation studies showed an increased number of CaV2.2 at the cell surface in CRMP-2-overexpressing neurons. These neurons also exhibited a significant increase in vesicular release in response to a depolarizing stimulus. Depolarization of CRMP-2-enhanced green fluorescent protein-overexpressing neurons elicited a significant increase in release of glutamate compared with control neurons. Toxin block of Ca(2+) entry via CaV2.2 abolished this stimulated release. Thus, the CRMP-2-Ca(2+) channel interaction represents a novel mechanism for modulation of Ca(2+) influx into nerve terminals and, hence, of synaptic strength.

Synaptic vesicles (SVs) are triggered to fuse with the surface membrane at the presynaptic transmitter release site (TRSs) core by Ca2+ influx through nearby attached CaV2.2 channels [see accompanying paper: Khanna et al. (2007)Eur. J. Neurosci., 26, 547-559] and are then recovered by endocytosis. In this study we test the hypothesis that the TRS core is linked to an endocytosis-related protein complex. This was tested by immunostaining analysis of the chick ciliary ganglion calyx presynaptic terminal and biochemical analysis of synaptosome lysate, using CaV2.2 as a marker for the TRS. We noted that CaV2.2 clusters abut heavy-chain (H)-clathrin patches at the transmitter release face. Quantitative coimmunostaining analysis (ICA/ICQ method) demonstrated a strong covariance of release-face CaV2.2 staining with that for the AP180 and intersectin endocytosis adaptor proteins, and a moderate covariance with H- or light-chain (L)-clathrin and dynamin coat proteins, consistent with a multimolecular complex. This was supported by coprecipitation of these proteins with CaV2.2 from brain synaptosome lysate. Interestingly, the channel neither colocalized nor coprecipitated with the endocytosis cargo-capturing adaptor AP2, even though this protein both colocalized and coprecipitated with H-clathrin. Fractional recovery analysis of the immunoprecipitated CaV2.2 complex by exposure to high NaCl (approximately 1 m) indicated that AP180 and S-intersectin adaptors are tightly bound to CaV2.2 while L-intersectin, H- and L-clathrin and dynamin form a less tightly linked subcomplex. Our results are consistent with two distinct clathrin endocytosis complexes: an AP2-containing, remote, non-TRS complex and a specialised, AP2-lacking, TRS-associated subcomplex linked via a molecular bridge. The most probable role of this subcomplex is to facilitate SV recovery after transmitter release.

CaV2.2 channels play a key role in the gating of transmitter release sites (TRS) at presynaptic terminals. Physiological studies predict that the channels are linked directly to the TRS but the molecular composition of this complex remains poorly understood. We have used a high-affinity anti-CaV2.2 antibody, Ab571, to test a range of proteins known to contribute to TRS function for both an association in situ and a link in vitro. CaV2.2 clusters were isolated intact on immunoprecipitation beads and coprecipitated with a number of these proteins. Quantitative staining covariance analysis (ICA/ICQ method) was applied to the transmitter release face of the giant calyx terminal in the chick ciliary ganglion to test for TRS proteins with staining intensities that covary in situ with CaV2.2, resulting in a covariance sequence of NSF>RIM>spectrin>Munc18>VAMP>alpha-catenin, CASK>SV2>Na+-K+ approximately 0. A high-NaCl dissociation challenge applied to the immunoprecipitated complex, using the fractional recovery (FR) method [Khanna, R., Li, Q. & Stanley, E.F. (2006) PLoS.ONE., 1, e67], was used to test which proteins were most intimately associated with the channel, generating an FR sequence for CaV2.2 of: VAMP>or=actin>tubulin, NSF, Munc18, syntaxin 1>spectrin>CASK, SNAP25>RIM, Na+-K+ pump, v-ATPase, beta-catenin approximately 0. Proteins associated with endocytosis are considered in a companion paper [Khanna et al. (2007)Eur. J. Neurosci., 26, 560-574]. With the exception of VAMP and RIM, the ICQ and FR sequences were consistent, suggesting that proteins that covary the most strongly with CaV2.2 in situ are also the most intimately attached. Our findings suggest that the CaV2.2 cluster is an integral element of a multimolecular vesicle-fusion module that forms the core of a multifunctional TRS.