F A C U L T Y   P R O F I L E 

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COLECRAFT, HENRY M, Ph.D.
Associate Professor of Physiology & Cellular Biophysics

Molecular Physiology of Voltage-Dependent Calcium Channels

Office: Russ Berrie Pavilion| 5th floor | Room 504
Telephone: 212.851.5372
Fax: 212.851.5375
Email:
hc2405@cumc.columbia.edu

Current Research
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.


Ion Channel Modulation in Health and Disease

Electrical signals (or action potentials) generated by ions traversing cell surface ion channels are a vital signaling mechanism in biology. Electrical signals co-ordinate the activity of millions of cardiac myocytes to generate the heartbeat; underlie the orchestrated firing of neurons that enable sight, speech, movement, and formation of memories; and control the release of hormones that control glucose homeostasis, growth, and development. Remarkably, these very diverse biological phenomena utilize a common signal transduction paradigm─ membrane depolarization encoded in action potentials leads to the opening of voltage-dependent Ca2+ (CaV) channels, permitting an influx of Ca2+ ions that trigger the appropriate biological response by binding specific Ca2+-sensitive proteins. Given their central role in converting electrical signals into biological responses it is no wonder that modulating the activity of CaV channels is a powerful method to regulate physiology, and is an important therapy for many serious diseases.

The lab is broadly interested in the molecular physiology and regulation of voltage-gated ion channels in health and disease. Active research is ongoing in the following areas:


1. Design, Development, and Applications of Novel Genetically Encoded Calcium Channel Blockers

Voltage-dependent calcium (CaV1/CaV2) channel dysfunction underlies many serious disorders including autism, migraines, pain, ataxias, neurodegenerative diseases, hypertension, and cardiac arrhythmias. In the nervous system, selective (CaV1/CaV2) channel blockers are sought after as potential therapeutics for stroke, neuropathic pain, psychiatric disorders, and Parkinson’s disease. Conventional (CaV1/CaV2) channel blockers are small organic molecules or toxins that interact with extracellular portions of pore-forming α1 subunits and either obstruct the channel pore or modulate gating. Such traditional (CaV1/CaV2) channel blockers have several limitations that significantly impede their therapeutic use for treating nervous system-related diseases: (1) they cannot be easily targeted to a defined neuronal population within a living organism, (2) there are no molecules that can effectively distinguish among the distinct L-type (CaV1.1 – CaV1.4) channels, and (3) they cannot discriminate among (CaV1/CaV2) channels either on the basis of the identity of their associated proteins, or their sub-cellular localization. We are interested in developing genetically-encoded CaV channel inhibitors to address these gaps. To that end, we have recently discovered that diverse cytosolic proteins which bind pore-forming α1-subunit intracellular loops can be converted into CaV channel blockers with tunable selectivity, kinetics and potency simply by anchoring them to the plasma membrane (Yang et al, 2007, Nature Chem. Biol. 3:795-804; Yang et al, 2013, Nature Commun. 4:2540. doi:10.1038/ncomms3540). We term this method, “channel inactivation induced by membrane-tethering an associated protein” (ChIMP). In this research program, we seek to build on the ChIMP method to develop novel genetically-encoded CCBs that permit inhibition of CaV channels with exquisite molecular and sub-cellular specificity.

 

2. Regulation of Calcium Channel Trafficking and Modulation in Heart

Ca2+ flowing through voltage-dependent L-type ( CaV1.2) channels into cardiac myocytes is a multi-dimensional signaling molecule that mediates excitation-contraction (EC) coupling, controls action potential duration, and regulates gene expression. Proper surface targeting of CaV1.2 and their basal/hormone-regulated activity is vital for normal cardiac physiology. CaV1.2 in heart associates with large supramolecular complexes that regulate channel trafficking, localization, turnover, and function. Beyond the primary CaV1.2 subunits (α1C, β, α2δ1 and γ) the complex includes (but is not limited to) calmodulin, kinases, phosphatases, scaffold proteins, BIN1, caveolin-3, and β-adrenergic receptors. Abnormal CaV1.2 function caused by mutations, defective trafficking, and/or altered formation of the macromolecular complex leads to pathological cardiac hypertrophy, heart failure, and life-threatening arrhythmias. Much of the prevailing dogma regarding mechanisms underlying CaV1.2 trafficking and modulation derives from studies on recombinant channels reconstituted in heterologous cells. Though this approach is useful and has yielded important insights, a serious limitation is that heterologous cells lack the complex cytoarchitecture and intracellular milieu of adult cardiomyocytes. Indeed, recent knock-in mice data indicate several well-accepted “facts” about CaV1.2 regulation derived from heterologous expression studies are not replicated in native heart, emphasizing the critical need for mechanistic studies in the context of actual cardiomyocytes. We have developed an innovative split-intein-mediated protein ligation approach to express informative CaV1.2 α1C mutants in cardiomyocytes (Subramanyam et al, 2013, Proc. Natl. Acad. Sci. 110:15461-6). This strategy circumvents the need to generate viruses encoding the entire α1C, which is technically challenging due to the large insert size of this protein. Compared to knock-in mice models, the split-intein approach is cost-effective and rapid, significantly increasing experimental throughput. Using this, our goals are to address longstanding questions regarding fundamental structure-function mechanisms underlying CaV1.2 trafficking, function, and modulation in heart cells. Questions include addressing molecular mechanisms of how CaV1.2 channels are targeted to dyads and caveolae; the role of various elements of the macromolecular complex in CaV1.2 trafficking and function in cardiomyocytes; how disease-causing mutations in CaV1.2 affect channel trafficking and modulation.

 

3. Molecular Regulation of Potassium Channel Complexes in Heart.

In human heart the slowly activating delayed rectifier K+ current IKS is critical for normal cardiac action potential repolarization. The IKS channel complex contains four pore-forming KCNQ1 (or Kv7.1) subunits assembled with two to four auxiliary KCNE1 peptides. Inherited loss-of-function mutations in KCNQ1 decrease IKs, leading to prolonged cardiac ventricular action potential duration, and long QT syndrome type 1 (LQT1). LQT1 is the most common inherited form of long QT syndrome (LQTS), a condition with high incidence (1 in 2000 live births), which predisposes to lethal ventricular arrhythmias that lead to syncope and sudden cardiac death (SCD). LQTS accounts for a significant portion of ~400,000 cases of SCD in the United States each year affecting all age groups from infants to the elderly. Present treatment options for LQTS, β-blocker therapy, implantable cardioverter defibrillators, and left cardiac sympathetic denervation, do not correct the underlying repolarization abnormality and all have significant limitations. Over a hundred different LQT1-causing mutations have been identified in KCNQ1, and because the mechanisms underlying IKS suppression likely vary for distinct mutations, this poses a challenge for devising effective new therapies. Variable penetrance and inconsistent correlation between severity of biophysical defects measured in vitro and risk of ventricular arrhythmias in patients further complicate management of the disease. Understanding how distinct LQT1 mutations suppress IKS requires a comprehensive view of their impact on channel biophysics, surface density, and dominant negative capacity. Elucidating LQT1 mechanisms across diverse mutations is needed to identify possible common mechanistic principles to streamline development of new therapies. Determining the functional impact of LQT1 mutations directly in cardiomyocytes may be more predictive of disease penetrance in humans and aid in risk stratification for management of the disease. Our long term objective is to define the precise molecular mechanisms underlying suppressed IKs across a spectrum of LQT1 mutations, and to bridge the mechanistic insights to advance personalized therapy for LQTS and life-threatening cardiac arrhythmias. Innovations made to advance these objectives include developing methods to quantitatively measure KCNQ1 channel surface density/trafficking in live cells with quantum dots, and establishing cultured adult cardiomyocyte model systems to elucidate LQT1 mechanisms in heart cells (Aromolaran et al, 2014, Cardiovasc. Res.104:501-511).


Selected Publications

Liu N, Yang Y, Ge L, Liu M, Colecraft HM, Liu X. 2017. Cooperative and acute inhibition by multiple C-terminal motifs of L-type Ca2+ channels. Elife. Jan 6;6. pii: e21989. doi:

Puckerin AA, Chang DD, Subramanyam P, Colecraft HM. 2016. Similar molecular determinants on Rem mediate two distinct modes of inhibition of CaV1.2 channels. Channels. 10(5):379-394.

Geng Y, Mosyak L, Kurinov I, Zuo H, Sturchler E, Cheng TC, Subramanyam P, Brown AP, Brennan SC, Mun HC, Bush M, Chen Y, Nguyen TX, Cao B, Chang DD, Quick M, Conigrave AD, Colecraft HM, McDonald P, Fan QR. 2016. Structural mechanism of ligand activation in human calcium-sensing receptor. Elife. Jul 19;5. pii: e13662. doi: 10.7554/eLife.13662.

Puckerin A, Aromolaran KA, Chang DD, Zukin RS, Colecraft HM, Boutjdir M, Aromolaran AS. 2016. hERG 1a LQT2 C-terminus truncation mutants display hERG 1b-dependent dominant negative mechanisms. Heart Rhythm. pii: S1547-5271(16)00057-6.

Chang, D.D. and Colecraft, H.M. 2015. Rad and Rem are non-canonical G-proteins with respect to the regulatory role of guanine nucleotide binding in CaV1.2 channel regulation. J. Physiol. 593:5075-5090.

Aromolaran, A., Chang, D., Kobertz, W.R., and Colecraft, H.M. 2014. KCNQ1 C-terminus assembly domain suppress IKs using different mechanisms. Cardiovasc. Res. 104(3):501-11.

Yang, T., Hendrickson, W., and Colecraft, H.M. 2014. Preassociated apocalmodulin mediates Ca2+-dependent sensitization of activation and inactivation of TMEM16A/B Ca2+-gated Cl─ channels. Proc. Natl. Acad. Sci. 111(51):18213-8.

Yang, T., Liu, Q., Kloss, B., Bruni, R., Kalathur, R.C., Guo, Y., Kloppmann, E., Rost, B., Colecraft, H.M., and Hendrickson, W.A., 2014. Structure and selectivity in bestrophin ion channels. Science, 346(6207):355-9.

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 o CaV β-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.