F A C U L T Y P R O F I L E
COLECRAFT, HENRY M, Ph.D.
Molecular Physiology of Voltage-Dependent Calcium Channels
Office: Russ Berrie Pavilion| 5th floor | Room 504
Molecular Physiology of Voltage-Dependent Calcium Channels.
Dr. Colecraft works on voltage-gated calcium channels which are critical for the biology of electrically active cells such as heart muscle and nerve cells. He focuses on fundamental understanding of how these channels operate in both normal and disease states, and engineering new molecules to regulate their activity for potential therapeutic benefit.
Structure-function mechanisms of CaV channel beta subunits
Voltage-dependent calcium (CaV) channels are multi-subunit protein complexes comprised of a main pore-forming a1 subunit and associated accessory proteins. Of the accessory proteins, b subunits (CaVb) are arguably the most important, being necessary for targeting the channel to the membrane and normalizing channel gating (the manner in which the channel opens and closes). Their essential role is emphasized by the severe neurological and cardiovascular phenotypes ensuing from CaVb dysregulation: epilepsy, altered threshold to pain, night blindness, and defects in cardiac development. We aim to understand: (1) at a quantitative level the mechanisms by which CaVbs exert such powerful effects on CaV channels; (2) the role of the a1-b interaction in disease; and (3) the potential of the a1-b association as a target for therapeutic drugs. A powerful complement of approaches is used including: whole-cell and single-channel patch clamp electrophysiology, fluorescence resonance energy transfer (FRET) determination of protein-protein interactions, molecular biology, bioinformatics, and modeling.
Design of novel genetically-encoded CaV channel inhibitors
Blockade of CaV channels is a therapeutic strategy for an impressive array of
cardiovascular and neurological diseases including: angina, hypertension, stroke, migraines, neuropathic pain, and epilepsy. In the quest for more effective and selective CaV channel blockers, the CaV channel a1-b subunit interaction is recognized as a promising locus, although attempts to target this site in the past have met with only limited success. It was recently discovered that the Rem/Rad/Gem/Kir (RGK) sub-family of Ras-like GTPases potently inhibit CaV channels by interaction with CaVb subunits. How do
these 'natural genetically-encoded CaV channel blockers' work? Can we learn from Nature's design to create a new generation of custom CaV channel blockers? We are taking cues from this remarkable protein family to create useful new genetically encoded CaV channel blockers for various customized applications.
Reverse engineering CaV channels in heart and neurons
CaV channels are central to biological function in heart and nerve cells, being necessary for generating the heartbeat, nerve cell communications, and regulation of gene expression. In the complicated cellular environment of heart cells and neurons, the mechanistic bases of CaV channel functions are often obscure, akin to a biological 'black box'. To elucidate mysterious aspects of CaV channel behavior in such native cells we employ a reverse engineering approach where viral-mediated gene delivery permits
selective genetic manipulation of CaV channel properties in heart and neuronal cells. Questions we are interested in addressing include: How are different CaV channels targeted to spatially distinct sites in heart and neurons? What mechanisms underlie CaV channel regulation by messenger molecules in heart and neurons? To address these, we combine viral-mediated gene delivery, quantitative confocal imaging, and electrophysiological approaches.
Subramanyam, P., Chang, D., Fang, K., Xie, W., Marks, A.R. and Colecraft, H.M. 2013. Manipulating L-type calcium channels in cardiomyocytes using split-intein–mediated protein trans-splicing. Proc. Natl. Acad. Sci. 110:15461-6. [Faculty of 1000, Recommended].
Yang, T., He, L.L., Chen, M.C., Fang, K., and Colecraft, H.M. 2013 Bio-inspired voltage-dependent calcium channel blockers. Nature Communications 4:2540. doi:10.1038/ncomms3540.
Shaw, R.M. and Colecraft, H.M. 2013. L-type calcium channel trafficking and local signaling in cardiac myocytes. Cardiovasc. Res. 98(2):177-86.
Yang, T., and Colecraft, H.M. 2013. Regulation of voltage-dependent calcium channels by RGK proteins. Biochim. Biophys. Acta. 1828(7):1644-54.
Yang, T., Puckerin, A., and Colecraft, H.M. 2012. Distinct RGK GTPases differentially use a1- and auxiliary b-binding-dependent mechanisms to inhibit CaV1.2/CaV2.2 channels. PLoS One, 7:e37079.
Fang, K., and Colecraft, H.M. 2011. Mechanism of auxiliary b-subunit-mediated trafficking of L-type (CaV1.2) channels. J. Physiol. 589:4437-4455.
Xu, X., Marx, S.O., and Colecraft, H.M. 2010. Molecular mechanisms, and selective pharmacological rescue, of Rem inhibited CaV1.2 channels in heart. Circ. Res. 107:620-630.
Yang, T., Xu, X., Kernan, T., Wu, V., and Colecraft, H.M. 2010. Rem, a member of the RGK GTPases, inhibits recombinant CaV1.2 channels using multiple mechanisms that require distinct conformations of the GTPase. J. Physiol. 588:1665-1681 (Cover article).
Xu, X., and Colecraft, H.M. 2009. Engineering Proteins for Custom Inhibition of CaV Channels. Physiology 24:210-218
Miriyala, J., Nguyen, T.K., Yue, D.T., and Colecraft, H.M. 2008. Role of CaVb subunits, and lack of functional reserve, in protein kinase A modulation of cardiac CaV1.2 channels. Circ. Res. 102:e54-e64.
Yang, T., Suhail, Y., Dalton, S., Kernan, T., and Colecraft, H.M. 2007. Genetically Encoded Molecules for Inducibly Inactivating CaV Channels. Nature Chem. Bio., In press.
Takahashi, S.X., Miriyala, J., Tay, L.H., Yue, D.T., and Colecraft, H.M. 2005. A CaVb SH3-GK interaction regulates multiple properties of voltage-gated Ca2+ channels. J. Gen. Physiol., 126:365-377.
Dalton, S., Takahashi, S.X., Miriyala, J., and Colecraft, H.M. 2005. A single CaVb can reconstitute both trafficking and macroscopic conductance of voltage-dependent calcium channels. J. Physiol., 567:757-769.
Agler, H.L., Evans, J., Tay, L-H., Anderson, M.J., Colecraft, H.M., and Yue D.T. 2005. G-protein gated inhibitory module of N-type (CaV2.2) Ca2+ channels. Neuron, 46:891-904.
Takahashi, S.X., Miriyala, J., and Colecraft, H.M. 2004. Membrane-associated guanylate kinase-like properties of b-subunits required for modulation of voltage-dependent Ca2+ channels. Proc. Natl. Acad. Sci. 101: 7193-98.
Takahashi, S.X., Mittman, S., and Colecraft, H.M. 2003. Distinctive modulatory properties of five human auxiliary b2 subunit splice variants on L-type Ca2+ channel gating. Biophys. J., 84:3007-3021.
Alseikhan, B.A., DeMaria, C.D., Colecraft, H.M., and Yue, D.T. 2002. Engineered calmodulins reveal the unexpected eminence of Ca2+ channel inactivation in controlling heart excitation. Proc. Natl. Acad. Sci. 99:17185-90.
Colecraft, H.M., Alseikhan B.A., Takahashi, S.X., Chaudhury, D, Mittman, S., Yegnasubramanian, V., Alvania, R.S., Johns, D.C., Marban, E., and Yue, D.T. 2002. Novel functional properties of Ca2+ channel b subunits revealed by their expression in adult heart cells. J. Physiol. 541:435-452.