Rajesh Khanna
Professor, Anesthesiology
Professor, BIO5 Institute
Professor, Neuroscience - GIDP
Professor, Pharmacology
Primary Department
Department Affiliations
(520) 626-4281
Work Summary
The focus of my laboratory’s’ research is to understand how ion channels, specifically, voltage-gated calcium and sodium channels, are regulated by novel protein interactions. Recent studies in my laboratory have focused on targeting protein-protein interactions with biologics (peptide aptamers) and small molecules; testing the activity of these novel chemical entities in biochemical and immunofluorescent-based assays of trafficking; examining their protein interaction signatures; testing them with whole cell voltage-clamp electrophysiology and voltage- and calcium sensitive fluorescence-based imaging. Regulating these protein networks to modulate the activity of ion channels in neurodegenerative diseases (Chronic Pain, Migraine, and Neurofibromatosis) is a key focus of the laboratory.
Research Interest
Regulation of Trafficking and Functions of Voltage-Gated Sodium and Calcium Channels; Identification of Novel Protein Regulators of Ion channels; Approaches to Targeting the Ion Channel Complexes in Neuropathic Pain and Neurodegenerative Diseases; Discovery of Novel Biologics and Small Molecules Targeting Pain and Neurodegenerative Diseases

Publications

Brittain, J. M., Wang, Y., Eruvwetere, O., & Khanna, R. (2012). Cdk5-mediated phosphorylation of CRMP-2 enhances its interaction with CaV2.2. FEBS letters, 586(21), 3813-8.

The axon/dendrite specification collapsin response mediator protein-2 (CRMP-2) bidirectionally regulates N-type voltage-gated Ca(2+) channels (CaV2.2). But how cyclin dependent kinase 5 (Cdk5)-mediated phosphorylation of CRMP-2 affects its interaction/regulation with CaV2.2 is unknown. CRMP-2-mediated enhancement of currents via CaV2.2 was not observed with a Cdk5 phospho-null CRMP-2-S522A mutant or in cells expressing an inactive Cdk5. Concomitant knockdown of endogenous CRMP2 and overexpression of CRMP2-S522A mutant refractory to knockdown phenocopied the reduction in Ca(2+) influx while the Rho kinase CRMP2-T555A mutant was ineffective. Cdk5-phosphorylated CRMP-2 had increased association with CaV2.2. These results identify an important role for Cdk5 in CRMP2-mediated CaV2.2 regulation.

Khanna, R., Li, Q., Schlichter, L. C., & Stanley, E. F. (2007). The transmitter release-site CaV2.2 channel cluster is linked to an endocytosis coat protein complex. The European journal of neuroscience, 26(3), 560-74.

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.

Moutal, A., Dustrude, E. T., Largent-Milnes, T. M., Vanderah, T. W., Khanna, M., & Khanna, R. (2017). Blocking CRMP2 SUMOylation reverses neuropathic pain. Molecular psychiatry.
BIO5 Collaborators
Rajesh Khanna, Tally M Largent-Milnes
Moutal, A., Yang, X., Li, W., Gilbraith, K. B., Luo, S., Cai, S., François-Moutal, L., Chew, L. A., Yeon, S. K., Bellampalli, S. S., Qu, C., Xie, J. Y., Ibrahim, M. M., Khanna, M., Park, K. D., Porreca, F., & Khanna, R. (2017). CRISPR/Cas9 editing of Nf1 gene identifies CRMP2 as a therapeutic target in neurofibromatosis type 1-related pain that is reversed by (S)-Lacosamide. Pain, 158(12), 2301-2319.

Neurofibromatosis type 1 (NF1) is a rare autosomal dominant disease linked to mutations of the Nf1 gene. Patients with NF1 commonly experience severe pain. Studies on mice with Nf1 haploinsufficiency have been instructive in identifying sensitization of ion channels as a possible cause underlying the heightened pain suffered by patients with NF1. However, behavioral assessments of Nf1 mice have led to uncertain conclusions about the potential causal role of Nf1 in pain. We used the clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (CRISPR/Cas9) genome editing system to create and mechanistically characterize a novel rat model of NF1-related pain. Targeted intrathecal delivery of guide RNA/Cas9 nuclease plasmid in combination with a cationic polymer was used to generate allele-specific C-terminal truncation of neurofibromin, the protein encoded by the Nf1 gene. Rats with truncation of neurofibromin, showed increases in voltage-gated calcium (specifically N-type or CaV2.2) and voltage-gated sodium (particularly tetrodotoxin-sensitive) currents in dorsal root ganglion neurons. These gains-of-function resulted in increased nociceptor excitability and behavioral hyperalgesia. The cytosolic regulatory protein collapsin response mediator protein 2 (CRMP2) regulates activity of these channels, and also binds to the targeted C-terminus of neurofibromin in a tripartite complex, suggesting a possible mechanism underlying NF1 pain. Prevention of CRMP2 phosphorylation with (S)-lacosamide resulted in normalization of channel current densities, excitability, as well as of hyperalgesia following CRISPR/Cas9 truncation of neurofibromin. These studies reveal the protein partners that drive NF1 pain and suggest that CRMP2 is a key target for therapeutic intervention.

Moutal, A., Honnorat, J., Massoma, P., Desormeaux, P., Bertrand, C., Malleval, C., Watrin, C., Chounlamountri, N., Mayeur, M., Besancon, R., Naudet, N., Magadoux, L., Khanna, R., Ducray, F., Meyronet, D., & Thomasset, N. (2015). CRMP5 Controls Glioblastoma Cell Proliferation and Survival through Notch-Dependent Signaling. CANCER RESEARCH, 75(17), 3519-3528.