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David Clapham, MD, PhD

Professor of Neurobiology
Harvard Medical School
Director, Basic Cardiovascular Research
Children’s Hospital
Department of Cardiology

MRRC Project(s)

R01 HL53483-06
G-Protein Gating of the Inward Rectifier K+ Channel

Ion channel biology lies at the core of neuronal activity. The research carried out in this laboratory relates to the most common mechanism of signal transduction. Additionally a new family of K+ selective channels has been discovered in a region of brain important for memory.

Research Description

Major Results

TRP Channels
TRP (transient receptor potential) ion channels, initially discovered in fruit flies as mediating vision, have recently been revealed in mammalian cells. Mammals have at least 28 distinct genes encoding different types of TRP channels, whose functions are just beginning to be understood. TRP channels are the vanguard of our sensory systems, responding to temperature, touch, pain, osmolarity, pheromones, taste and other stimuli. But their role is much broader than sensory. They are an ancient sensory apparatus for the cell, not just the multicellular organism, and they have been adapted to respond to all manner of stimuli, from both within and without the cell.

Transient Receptor Potential (TRP) channels were first described in Drosophila, where photoreceptors carrying trp gene mutations exhibited a transient voltage response to continuous light. Unlike most ion channels, TRP channels are identified by their homology because their functions are disparate and often unknown. Their known functions are diverse. Yeast use a TRP channel to perceive and respond to hypertonicity. Nematodes use TRP channels at the tips of neuronal dendrites in their "noses" to detect and avoid noxious chemicals. Male mice use a pheromone-sensing TRP channel to tell males from females - they mate indiscriminately when it is inactive. Humans use TRP channels to appreciate sweet, bitter and umami tastes, and to discriminate warmth, heat and cold. In each of these cases, TRPs mediate sensory transduction, not only in a classic sense, for the entire multicellular organism, but also at the level of single cells.

We cloned and characterized several of the mammalian TRP channels. TRPM7 is a novel protein that is both an ion channel and a protein that phosphorylates other proteins. This channel/kinase protein is present in all cells and is permeant to Ca2+. The kinase domain of TRPM7 directly binds phospholipase C, a common cell-signaling enzyme. G protein-linked or growth factor receptors that activate phospholipase C potently inhibit channel activity. We are currently testing the idea that TRPM7 plays an important role in cell shape.

Our laboratory identified a member of the vanilloid channel family, human TRPV3 (that is expressed in skin, tongue, dorsal root ganglion, trigeminal ganglion, spinal cord and brain. Increasing temperature from 22o8C to 40oC in mammalian cells elevated intracellular Ca2+ by activating a nonselective cationic conductance. As in found in sensory neurons, the current is steeply dependent on temperature, sensitized with repeated heating, and displays a marked hysteresis on heating and cooling. Thus TRPV3 is a precise detector of changes in temperature.

Classical mammalian TRP channels (TRPC) can combine with each other to perform unique functions. We demonstrated that TRPC1 and TRPC5 join to form a neuronal channel present in the hippocampus, cortex, and amygdala. TRPC1/TRPC5 channels were activated by GTP-binding protein Gq-coupled receptors but not by depletion of intracellular Ca2+ stores. TRPC5, without TRPC1, is in growth cones of young rat hippocampal neurons. We found that TRPC5 channels are important components of the mechanism controlling neurite extension and growth cone motility.

New calcium-selective channels.
Calcium and cyclic nucleotides have are crucial elements in mammalian fertilization, but the channels comprising the Ca2+-permeation pathway in sperm motility are poorly understood. We found a sperm-specific cation channel (CatSper, for Cation channel of Sperm), whose amino-acid sequence most closely resembles a single, six-transmembrane-spanning unit of the voltage-dependent Ca2+-channel four domain structure. CatSper was only present in the principal piece of the sperm tail. Disruption of the CatSper gene resulted in male sterility in otherwise normal mice. Sperm motility was decreased in mice lacking the CatSper gene, and their sperm were unable to fertilize intact eggs. We have identified two other genes that encode related channels and genetically targeted mice should soon reveal their function in fertility. CatSper may be an excellent target for non-hormonal contraceptives for both men and women.

The pore-forming subunits of the well-known voltage-gated sodium- and Ca2+-selective channels are made up of 4 repeated domains of 6-transmembrane segments. We discovered an ion channel (NaChBac) in the extremophile bacteria, Bacillus halodurans, that was encoded by only one such segment. The amino acid sequence of the channel suggested that it was selective for Ca2+, and like voltage-gated Ca2+ channels, was activated by voltage and blocked by Ca2+ channel drugs. However, the channel was selective for sodium. The identification of this simple channel will help us understand how more complex mammalian voltage-gated cation-selective channels accomplish their function. We are currently trying to obtain the high-resolution structure of this basic unit of ion channels by X-ray crystallography. This structural information will help us understand how the sodium channels controlling excitability in humans select for sodium over other ions, and how voltage-gated channels sense voltage changes to open the channel. We have also found Ca2+-selective channels from bacteria that should help us understand how these channels allow only Ca2+ through their pores.

During intracellular Ca2+ signaling mitochondria accumulate significant amounts of Ca2+ from the cytosol. Mitochondrial Ca2+ uptake controls the rate of energy production, shapes the amplitude and spatio-temporal patterns of intracellular Ca2+ signals, and is instrumental to cell death. This Ca2+ uptake is via the mitochondrial Ca2+ uniporter (MCU) located in the organelle's inner membrane. Up to now it has been unclear whether the MCU is a carrier or a channel. By patch-clamping the inner mitochondrial membrane, we identified the MCU as a novel, highly Ca2+-selective ion channel. This unique channel binds Ca2+ with extremely high affinity, enabling high Ca2+ selectivity despite relatively low cytoplasmic Ca2+ concentrations, and is especially effective for Ca2+ uptake into energized mitochondria.

In summary, the amount of Ca2+ in cells is controlled more tightly than any other ion in the body. Too much, or too little, Ca2+ inside the cell causes cell death. Cell receptors and signaling pathways thus closely guard where, when, and how Ca2+ is admitted through ion channels. Admitted Ca2+ has dramatic consequences for cell function at all levels of its activity; motility, proliferation, transcription of genes, growth, secretion, and contraction. By identifying and understanding these ion channels, drugs may be developed that alleviate many diseases.

Publications

Krapivinsky G, Gordon E, Wickman K, Velimirovic B, Krapivinsky L, and Clapham DE. The G protein-gated atrial K+ channel, IKACh, is a heteromultimer of two inwardly rectifying K+ channel proteins. Nature 1995; 374:135-141.

Perez-Terzic C, Pyle J, Jaconi M, Stehno-Bittel L, and Clapham DE. Conformational states of the nuclear pore complex induced by depletion of nuclear Ca2+ stores. Science, 1996; 273, 1875-1877.

Wickman K, Nemec J, Gendler S, and Clapham DE. Abnormal heart rate regulation in GIRK4 knockout mice. Neuron, 1998, 20, 103-114.

Krapivinsky G, Medina I, Eng L, Krapivinsky L, Yang Y, and Clapham DE A novel inward rectifier K+ channel with unique pore properties. Neuron, 1998, 20, 995-1005.

Schiller J, Schiller Y, and Clapham DE. The NMDA receptor channel amplifies calcium influx into dendritic spines during associative pre- and postsynaptic activation. Nature Neurosci 1998, 1, 114-118.

Arnold, D and Clapham DE. Molecular determinants for subcellular localization of PSD-95 with an interacting K+ Channel. Neuron, 1999, 23, 149-157.

Medina, I, Krapivinsky, GB, Krapivinsky, L, Arnold, S, Kovoor, P, and Clapham, DE. A switch mechanism for Gbgactivation of IKACh. J Biolog Chem, 2000, 275, 29709-29715.

Runnels, LW, Yue, L, and Clapham, DE. TRP-PLIK, a bifunctional protein with both kinase and ion channel activities. Science 2001.

Runnels LW, Yue L, Clapham DE. TRP-PLIK, a bifunctional protein with kinase and ion channel activities. Science 2001;291:1043-1047.

Stribing C, Krapivinsky G, Krapivinsky L, Clapham DE. TRPC1 and TRPC5 form a novel cation chennel in mammalian brain. Neuron 2001;29(29):645-655.

Yue L, Peng J-B, Hediger Ma, Clapham DE. CaT1 manifests the pore properties of the calcium-release-activated calcium channel. Nature 2001;410:705-709.

Corey S, Clapham DE. The stoichiometry of Gbg binding to G-protein-regulated inwardly rectifying K+ channels (GIRKs). The Journal of Biological Chemistry 2001;276:11409-11413.

Kovoor K, Wickman K, Maguire CT, Pu W, Gehrmann J, Berul CI, Clapham DE. Evaluation of the role of IKACh in atrial fibrillation using a mouse knockout model. J Am College of Cardiol 2001;27:2136-2143.

Hsu S, O'Connell PJ, Klyachko VA, Badminton MN, Thomson AW, Clapham DE, Jackson MB, Ahern GP. Fundamental Ca2+ signaling mechanisms in mouse dendritic cells: CRAC is the major CA2+ entry pathway. J Immunology 2001;166:6126-6133.

Ren D, Navarro B, Perez G, Jackson AC, Hsu S, Shi Q, Tilly JL, Clapham DE. A sperm ion channel required for sperm motility and male fertility. Nature 2001;413:603-609.

Quill TA, Ren D, Clapham DE, Garbers DL. A voltage-gagted ion channel expressed specifically in spermatoxoa. PNAS 2001;98:12527-12531.

Ren D, Navarro B, Xu H, Hue L, Shi Q, Clapham DE. A prokaryotic voltage-gated sodium channel. Science 2001;294:2372-2375.

Wickman K, Pu WT, Clapham DE. Structural characterization of the mouse Girk genes. Gene 2002:241-250.

Sandler VM, Wang S, Angelo K, Lo HG, Breakefield XO, Clapham DE. Modified herpes simplex virus delivery of enhanced GFP into the central nervous system. J Neurosci Meth 2002;121:211-219.

Runnels LW, Yue L, Clapham DE. The TRPM7 channel is inactivated by PIP2 hydrolysis. Nature Cell Biol 2002;10:329-336.

Xu H, Ramsey IS, Kotecha SA, Moran MM, Chong JA, Lawson D, Ge P, Lilly J, Silos-Santiago I, Xie Y, DiStefano PS, Curtis R, Clapham DE. TRPV3 is a calcium-permeable temperature-sensitive cation channel. Nature 2002;418:181-186.

Yue L, Navarro B, Ren D, Ramos A, Clapham DE. The cation selectivity filter of the bacterial sodium channel, NaChBac. J General Physiol 2002;120:845-853.

Roberson C, Clapham DE. G protein-coupled receptor signaling through ion channels. In: Davies CH, Pangalos MN, editors. GPCRs in the CNS - The GPCR Superfamily: Oxford Unviersity Press; 2002, 108-123.

Montell C, Birnbaumer L, Flockerzei V, Bindels RJ, Bruford EA, Caterina MA, Clapham DE, Harteneck C, Heller S, Julius D, Mori Y, Penner R, Prawittt D, Scharenberg AM, Schultz G, Shimizu N, Zhu M. A unified nomenclature for the superfamily of TRP cation channels. Molec Cell 2002;9:229-231.

Clapham DE. Sorting out MIC, TRP, and CRAC ion channels. J Gen Physiol 2002;120:217-220.

Wei X, Henke V, Strubing C, Clapham DE. Real time imaging of nuclear permeation by EGFP in single intact cells. Biophys J 2003;84:1317-1327.

Oancea E, Bezzerides VJ, Clapham DE. Protein Kinase D acts as a memory sensor to increase cellular motility. Dev Cell 2003;4:561-574.

Greka A, Navarro B, Oancea E, Duggan A, Clapham DE. TRPC5 is a regulator of hippocampal neurite length and growth cone morphology. Nature Neurosci 2003;6:837-845.

Strübing C, Krapivinsky G, Krapivinsky L, Clapham DE. Formation of novel TRPC channels by complex subunit interactions in embryonic brain. J Biol Chem 2003;278:39104-39019.

Krapivinsky G, Krapivinsky L, Manasian Y, Ivanov I, Tyzio R, Pellegrino C, Ben-Ari Y, Clapham DE, Medina I. The NMDA receptor is coupled to the ERK pathway by a specific interaction between NR2B and Ras-GRF1. Neuron 2003;40:775-784.

Carlson AE, Westenbroek RE, Quill T, Ren D, Clapham DE, Hille B, Garbers DL, Babcock DF. CatSper1 required for evoked Ca2+ entry and control of flagellar function in sperm. PNAS 2003;14864-14868.

Clapham DE, Montell C, Schultz G, Julius D. International Union of Pharmacology. XLIII. Compendium of voltage-gated ion channels: Transient receptor potential channels. In: Pharmacol Rev; 2003;591-596.

Clapham DE. TRP Channels as cellular sensors. Nature 2003;436:517-524.

Clapham DE. Symmetry, selectivity, and the 2003 Nobel Prize. Cell 2003;115:641-646.

Xu H, Jin J, DeFelice LJ, Andrews NC, Clapham DE. A spontaneous, recurrent mutation in divalent metal transporter-1 exposes a calcium entry pathway. Public Library of Science (PLOS) 2003, 2:378-386.

Kirichok Y, Krapivinsky G, Clapham DE. The mitochondrial calcium uniporter is a novel Ca2+-selective ion channel. Nature 2004;427:360-364.

Koishi R, Xu H, Ren D, Navarro B, Spiller BW, Shi Q, Clapham DE. A superfamily of voltage-gated sodium channels in bacteria. J Biol Chem 2004;279(10):9532-8.

Moran MM, Xu HX, Clapham DE. TRP ion channels in the nervous system. Curr Opin Neurobiol 2004, in press.

See Dr. Clapham's publications via PubMed

Contact Information

E-mail: David Clapham, MD, PhD
Professor of Neurobiology
Harvard Medical School
Director, Basic Cardiovascular Research
Children’s Hospital
Department of Cardiology