Henry Colecraft, PhD

Structure-Function Mechanisms of CaV Channel β Subunits

Voltage-dependent calcium (CaV) channels are multi-subunit protein complexes comprised of a main pore-forming α1 subunit and associated accessory proteins. Of the accessory proteins, β subunits (CaVβ) 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 CaVβ 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 CaVβs exert such powerful effects on CaV channels; (2) the role of the α1-β interaction in disease; and (3) the potential of the α1-β 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.