David Clapham, M.D., Ph.D.

 

Aldo R. Castaneda Professor of Cardiovascular Research

Boston Childrenís Hospital
Enders Building, Room 1309
320 Longwood Avenue
Boston, MA 02115
Tel: 617-919-2680
Fax: 617-731-0787
Email: dclapham@enders.tch.harvard.edu
Visit my lab page here and here.







Summary: Our research focuses on the control of intracellular calcium signaling by ion channels.

Organisms control intracellular Ca2+ more tightly than any other ion. Normal concentrations of free intracellular Ca2+ are 20,000-fold lower than extracellular concentrations; above a narrow intracellular range, cells die. Cell receptors and signaling pathways thus closely guard where, when, and how Ca2+ is admitted through ion channels. Once admitted, Ca2+ has dramatic consequences for cell function at all levels of its activity. We are actively investigating the following areas:

1.
Na+ and Ca2+ selective pores. We were surprised to find a large group of voltage-gated ion channels (NaChBac) in bacteria. Remarkably, the channel expresses well in mammalian cells where its structure and function can be examined in mutagenesis and patch clamp studies. The identification of this simple channel will help us understand how more-complex mammalian voltage-gated cation-selective channels accomplish their functions. In collaboration with the Yan lab, we studied the high-resolution structure of this basic unit of ion channels. This structural information will help us understand how the Na+ channels controlling excitability in humans. We have also found Ca2+-selective channels from bacteria that should help us understand how these channels allow only Ca2+ through their pores.

2.
TRP Channels. TRP channels are the vanguard of our sensory systems, responding to temperature, touch, pain, osmolarity, pheromones, taste, and other stimuli.

TRPC5 channel function in CNS plasticity and amygdala. We demonstrated that TRPC1 and TRPC5 join to form a neuronal channel present in the hippocampus, cortex, and amygdala. We found that TRPC5 channels are important components of the mechanism controlling neurite extension and growth cone motility. These channels are rapidly inserted into the plasma membrane upon growth factor stimulation. Potentiation results in TRPC5 channel activation preferentially during periods of repetitive firing, or coincident neurotransmitter receptor activation. We deleted the TRPC5 gene in mice and, working with the Bolshakov laboratory, found that these mice have reduced innate fear. Experiments in amygdalar brain slices showed that mutant mice exhibited significant reductions in responses mediated by synaptic activation of Group I metabotropic glutamate and cholecystokinin 2 receptors in neurons of the amygdala. These experiments provide genetic evidence that TRPC5, activated via G protein-coupled neuronal receptors, has an essential function in innate fear.

TRPV3 channel function in sensing. Our laboratory identified a member of the vanilloid channel family, human TRPV3, which is expressed in skin, spinal cord, and brain. Increasing temperature above 30oC in mammalian cells elevates intracellular Ca2+ by activating TRPV3. As is found in sensory neurons, the current is steeply dependent on temperature, sensitized with repeated heating, and displays a marked hysteresis on heating and cooling. We have identified plant compounds that activate TRPV3, TRPV1, and other TRP channels and are currently working on TRPV3 contributions to alterations in pain sensing, such as temperature allodynia.

Transient Receptor Potential-Melastatin-like 7 (Trpm7) encodes a protein that functions in all cells as both an ion channel and a kinase. Deletion of mouse Trpm7 disrupts embryonic development at a very early stage. TRPM7s function in synaptic vesicles of sympathetic neurons and forms molecular complexes with synaptic vesicle proteins. Currently we are studying how the channel and kinase functions are related, and why the channel is so crucial to embryonic development.

3.
Mitochondrial Ca2+ regulation. 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 spatiotemporal patterns of intracellular Ca2+ signals, and is instrumental to cell death. This Ca2+ uptake is primarily via the mitochondrial Ca2+ uniporter (MCU) located in the organelle's inner membrane. By patch-clamping the inner mitochondrial membrane, we identified the MCU as a novel, highly Ca2+-selective ion channel (MiCa; Mitochondria Ca channel). In parallel with the search for MiCa, we searched for genes that regulate mitochondrial Ca2+ and H+ levels using a genome-wide Drosophila RNAi screen. The mammalian homolog of one Drosophila gene identified in the screen was found to specifically mediate coupled Ca2+/H+ exchange. RNAi knockdown, overexpression, and liposome reconstitution of the purified protein demonstrate that it is a mitochondrial Ca2+/H+ antiporter. We are currently investigating its function on metabolism in Letm1 knockout mice, a model for Wolf-Hirschhorn syndrome-related seizures.

4.
Hv1, the voltage-gated proton channel. Cation channels belong to a large class of proteins conserved from bacteria to man termed the voltage gated-ligand superfamily. Each channel subunit is formed by a polypeptide chain of 6-transmembrane-spanning segments (S1-S6). S1-S4 functions as a self-contained voltage-sensing domain (VSD), in essence a positively charged handle that moves in response to voltage changes. The VSD transmits force via a linker to the S5-S6 pore domain, thereby opening or closing the channel. We discovered a mammalian VSD protein (Hv1) that lacks a discernable pore domain but is sufficient for expression of voltage-sensitive, proton-selective ion channel. Hv1 currents are activated at depolarizing voltages, sensitive to the transmembrane pH gradient, H+-selective, and Zn2+-sensitive. In phagocytic leukocytes, this channel is required to support the oxidative burst that underlies microbial killing by the innate immune system. Hv1 is present in microglia in brain, and mice lacking this channel are resistant to stroke damage since Hv1 support reactive oxygen species generation during ischemia by enabling NADPH oxidase function.

5.
Ion channels of spermatozoa. Calcium and cyclic nucleotides are crucial elements in mammalian fertilization, but the channels composing 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, 6-transmembrane-spanning unit of the voltage-dependent Ca2+ channel four-domain structure. CatSper1 is only present in the principal piece of the sperm tail. Disruption of the CatSper1 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 identified 3 other CatSper genes encoding ion channel subunits in sperm and demonstrated that the CatSper channel is a heterotetramer of CatSper1, 2, 3, and 4 proteins. Deletion of any one of the 4 CatSper genes in mice results in an identical male infertility phenotype due to loss of hyperactivated motility. In addition, we used purification, mass spectrometry, and functional studies to show that the channel complex contains 5 accessory subunits, making it the most complex of ion channels.

Making the first whole-sperm recordings of spermatozoa, we found that spermatozoa are unique in their physiology, having two major, sperm-specific ion channel currents that regulate motility. Spermatozoa encounter progressively more alkaline environments as they ascend the female reproductive tract. We recorded the ion currents from mature mouse sperm and found that CatSper current, one of two major currents in sperm, is highly activated by alkalinity. We also identified a novel sperm-specific potassium channel, called KSper, and showed that its properties are most similar to the mSlo3 K+ channel.


For a complete listing of publications click here.



Last Update: 11/7/2013