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


Sun, L., Li, Q., Khanna, R., Chan, A. W., Wong, F., & Stanley, E. F. (2006). Transmitter release face Ca2+ channel clusters persist at isolated presynaptic terminals. The European journal of neuroscience, 23(5), 1391-6.

Ca(2+) influx through N-type Ca(2+) channels (CaV2.2) is known to be critical for transmitter release at many synapses. These channels are known to be localized to transmitter release sites, but their anchoring mechanism remains unknown. Recent studies have demonstrated that presynaptic organization is subject to interactions with the postsynaptic cell or the intervening extracellular matrix. We used a previously described high-affinity antibody against the N-type Ca(2+) channels, Ab571, to localize Ca(2+) channel clusters at the release face of an isolated giant calyx-type synapse to test whether the maintenance of these clusters requires an intact extracellular matrix or contact with the postsynaptic cell. Because the number of Ca(2+) channel clusters was unchanged after extracellular matrix dispersal or nerve terminal isolation, we conclude that presynaptic transmitter release face Ca(2+) clusters can be maintained independently of extracellular influences. Our results suggest that a presynaptic molecular scaffold is responsible for the maintenance of release site Ca(2+) channel clusters.

Myers, M. P., Khanna, R., Lee, E. J., & Papazian, D. M. (2004). Voltage sensor mutations differentially target misfolded K+ channel subunits to proteasomal and non-proteasomal disposal pathways. FEBS letters, 568(1-3), 110-6.

In Shaker K(+) channels, formation of an electrostatic interaction between two charged residues, D316 and K374 in transmembrane segments S3 and S4, respectively, is a key step in voltage sensor biogenesis. Mutations D316K and K374E disrupt formation of the voltage sensor and lead to endoplasmic reticulum retention. We have now investigated the fates of these misfolded proteins. Both are significantly less stable than the wild-type protein. D316K is degraded by cytoplasmic proteasomes, whereas K374E is degraded by a lactacystin-insensitive, non-proteasomal pathway. Our results suggest that the D316K and K374E proteins are misfolded in recognizably different ways, an observation with implications for voltage sensor biogenesis.

Chan, A. W., Khanna, R., Li, Q., & Stanley, E. F. (2015). Munc18: a presynaptic transmitter release site N type (CaV2.2) calcium channel interacting protein. Channels (Austin, Tex.), 1(1), 11-20.

Munc18 is a presynaptic protein that is essential for transmitter release. Recent studies have indicated that this protein is involved in secretory vesicle docking but its binding partners in this role remain a mystery. We demonstrate using the isolated calyx-type presynaptic terminal of the chick ciliary ganglion that staining for Munc18 colocalizes and covaries with that for transmitter release site N type calcium channels (CaV2.2), consistent with elements of a common release site complex. Biochemical analysis demonstrated that the protein coprecipitates with CaV2.2 from lysates of rat or chick brain, including its synaptic, long-splice variant; presynaptic terminal surface membrane proteins, and a cell line coexpressing Munc18 and CaV2.2. Munc18 bound with high affinity to the CaV2.2 II-III intracellular loop, low affinity to the I-II loop but not to other channel intracellular regions. Over-expression of Munc18 in dorsal root ganglion neurons did not affect CaV2.2 current amplitude or fast kinetics but siRNA-knockdown resulted in a negative shift in the steady state inactivation curve, an effect attributed to an indirect action via syntaxin 1. Recombinant Munc18 also coprecipitated strongly with the v-SNARE synaptotagmin, but only weakly with other SNAREs. Thus, the calcium channel may serve as a surface membrane platform anchoring a Munc18-containing bridge to synaptotagmin and the synaptic vesicle.

Jugloff, D. G., Khanna, R., Schlichter, L. C., & Jones, O. T. (2000). Internalization of the Kv1.4 potassium channel is suppressed by clustering interactions with PSD-95. The Journal of biological chemistry, 275(2), 1357-64.

The contribution of voltage-dependent ion channels to nerve function depends upon their cell-surface distributions. Nevertheless, the mechanisms underlying channel localization are poorly understood. Two phenomena appear particularly important: the clustering of channels by membrane-associated guanylate kinases (MAGUKs), such as PSD-95, and the regional stabilization of cell-surface proteins by differential suppression of endocytosis. Could these phenomena be related? To test this possibility we examined the effect of PSD-95 on the internalization rate of Kv1.4 K(+) channels in transfected HEK293 cells using cell-surface biotinylation assays. When expressed alone Kv1.4 was internalized with a half-life of 87 min, but, in the presence of PSD-95, Kv1.4 internalization was completely suppressed. Immunochemistry and electrophysiology showed PSD-95 had little effect on total or cell-surface levels of Kv1.4 or on current amplitude, activation, or inactivation kinetics. Clustering was necessary and sufficient to suppress Kv1.4 internalization since C35S-PSD-95, a mutant reported to bind but not cluster Kv1.4, (confirmed by imaging cells co-expressing a functional, GFP-variant-tagged Kv1.4) restored and, surprisingly, enhanced the rate of Kv1.4 internalization (t((1)/(2)) = 16 min). These data argue PSD-95-mediated clustering suppresses Kv1.4 internalization and suggest a fundamentally new role for PSD-95, and perhaps other MAGUKs, orchestrating the stabilization of channels at the cell-surface.

Dustrude, E. T., Perez-Miller, S., François-Moutal, L., Moutal, A., Khanna, M., & Khanna, R. (2017). A single structurally conserved SUMOylation site in CRMP2 controls NaV1.7 function. Channels (Austin, Tex.), 11(4), 316-328.

The neuronal collapsin response mediator protein 2 (CRMP2) undergoes several posttranslational modifications that codify its functions. Most recently, CRMP2 SUMOylation (addition of small ubiquitin like modifier (SUMO)) was identified as a key regulatory step within a modification program that codes for CRMP2 interaction with, and trafficking of, voltage-gated sodium channel NaV1.7. In this paper, we illustrate the utility of combining sequence alignment within protein families with structural analysis to identify, from several putative SUMOylation sites, those that are most likely to be biologically relevant. Co-opting this principle to CRMP2, we demonstrate that, of 3 sites predicted to be SUMOylated in CRMP2, only the lysine 374 site is a SUMOylation client. A reduction in NaV1.7 currents was the corollary of the loss of CRMP2 SUMOylation at this site. A 1.78-Å-resolution crystal structure of mouse CRMP2 was solved using X-ray crystallography, revealing lysine 374 as buried within the CRMP2 tetramer interface but exposed in the monomer. Since CRMP2 SUMOylation is dependent on phosphorylation, we postulate that this state forces CRMP2 toward a monomer, exposing the SUMO site and consequently, resulting in constitutive regulation of NaV1.7.