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Summary: David Clapham is interested in the signal transduction control of ion channel activity and the role of calcium as an intracellular messenger.

The human genome contains more than 150 genes that encode ion channels brokering the passage of charged ions across impermeable lipid bilayers. While energy-requiring pumps labor to build charge and concentration gradients across the membrane, ion channels spend this stored energy, much as a switch releases the electrical energy of a battery. Small conformational changes cause channels to open, allowing ~10 million ions to flow per second. Normal concentrations of free intracellular Ca²⁺ are 20,000-fold lower than extracellular concentrations, and cells tightly regulate these intracellular levels. Excitable cells have been equipped with specialized voltage-sensitive and highly selective Ca²⁺ channels. Opening of these channels provides the sharp, decisive rise in Ca²⁺ required for rapid contraction in heart and skeletal muscle, as well as for exocytosis at nerve terminals. Nonexcitable cells, such as blood cells and cells of solid organs, use a distinct set of Ca²⁺-permeant channels. My laboratory is investigating the various ways Ca²⁺ entry into cells is controlled by newly discovered classes of ion channels.

TRP Channels
TRP (transient receptor potential) ion channels, initially discovered in fruit flies as mediators of 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.

	Recording from mature mouse sperm... Direct recordings from mitochondria... Sperm calcium change...

TRP channels were first described in Drosophila, where photoreceptors carrying trp gene mutations exhibit 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. TRPM7 appears to play a role in cell survival, and is activated under anoxic conditions in the brain.

Our laboratory identified a member of the vanilloid channel family, human TRPV3, which is expressed in skin, tongue, dorsal root ganglion, trigeminal ganglion, spinal cord, and brain. Increasing temperature from 22^oC to 40^oC in mammalian cells elevated intracellular Ca²⁺ by activating a nonselective cationic conductance. 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. Thus, TRPV3 is a precise detector of changes in temperature. We have recently identified plant compounds that activate TRPV3.

Classical mammalian TRP channels (TRPCs) 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. These channels are rapidly inserted into the plasma membrane upon growth factor stimulation.

New Calcium-Selective Channels
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, six-transmembrane-spanning unit of the voltage-dependent Ca²⁺ channel four-domain structure. CatSper is 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; genetically targeted mice should soon reveal the function of these channels in fertility. We recorded the ion currents from mature mouse sperm and found that CatSper current, one of two major currents in sperm, is pH sensitive. CatSper may be an excellent target for nonhormonal contraceptives for both men and women.

The pore-forming subunits of the well-known voltage-gated Na⁺- and Ca²⁺-selective channels are made up of four repeated domains of six-transmembrane segments. We discovered an ion channel (NaChBac) in the extremophile bacteria Bacillus halodurans that is encoded by only one such segment. The amino acid sequence of the channel suggested that it is selective for Ca²⁺, and like voltage-gated Ca²⁺ channels, is activated by voltage and blocked by Ca²⁺ channel drugs. However, the channel was selective for Na⁺. The identification of this simple channel will help us understand how more-complex mammalian voltage-gated cation-selective channels accomplish their function. We are 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 Na⁺ channels controlling excitability in humans select for Na⁺ 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. Recently, we identified a novel proton-selective ion channel that consists of only a voltage-sensor domain. An understanding of this domain will lead to a better understanding of ion channel gating.

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 via the mitochondrial Ca²⁺ uniporter (MCU) located in the organelle's inner membrane. Until 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. The identification and characterization of these ion channels may make it possible to develop drugs that alleviate many diseases.

Grants from the National Institutes of Health provided partial support for the work described above.

Last updated: November 17, 2006