Imagine building a four-mile-high dike around the deepest part of the ocean. This is analogous to what a cell does when it reduces calcium ions to 20,000-fold lower levels inside the cell than surrounding the cell. Uncontrolled Ca2+ leaks induce cell death, whereas controlled Ca2+ entry triggers an enormous array of actions, ranging from secretion to cell division. Ion channels are the electrical switches that control these actions. One ion channel directs the flow of ~10 million ions per second, in turn rapidly changing intracellular Ca2+ levels. The human genome contains more than 200 genes encoding ion channels, the cell's transistors. My laboratory finds and investigates ion channels that regulate Ca2+ signaling.
TRP (transient receptor potential) proteins are the second largest class of ion channels but are the least well understood. Mammals have at least 28 distinct genes encoding TRP channels. These are the vanguard of our sensory systems, responding to temperature, touch, pain, osmolarity, pheromones, taste, and other stimuli, but their roles are much broader than sensation. 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 outside the cell.
TRPM7, the ubiquitous chanzyme. Trpm7 encodes a protein that functions both as an ion channel and a kinase. Deletion of Trpm7, which is found in practically all cells of vertebrates, is lethal at an early embryonic stage. Thymocyte-specific deletions disrupt thymopoiesis, leading to a developmental block of thymocytes and a progressive depletion of thymic medullary cells. Although one pervasive theory maintains that TRPM7 regulates Mg2+ homeostasis, deletion of Trpm7 in T cells fails to affect either acute Mg2+ responses or total cellular Mg2+ levels. Widespread phenotypes in our other Cre lines suggest that the kinase is important in an early step in development of many cell lineages. This is the only ion channel gene demonstrated to have such a fundamental role in developmental processes, with implications for all aspects of vertebrate biology.
Canonical TRP channel function in the brain. TRPC1, C4, and C5 form neuronal channels in the hippocampus, cortex, and amygdala. TRPC5 channels affect neurite extension and growth cone motility, being rapidly inserted into the plasma membrane upon growth factor stimulation. Increasing intracellular Ca2+ potentiates TRPC5 during periods of repetitive firing or coincident neurotransmitter receptor activation. We deleted this gene in mice and found that these mice have reduced innate fear. Experiments in amygdalar brain slices showed that mutant mice exhibit significant reductions in responses mediated by synaptic activation of metabotropic glutamate and cholecystokinin 2 receptors in neurons of the amygdala.
The mucolipins: channels of intracellular organelles. TRPML1–3 are ion channels in intracellular endosomes and lysosomes. Mutations in human TRPML1 result in mucolipidosis type IV, a severe inherited neurodegenerative disease associated with defective lysosomal biogenesis and trafficking. An activating mutation in TRPML3 causes Ca2+ overload and cell death, resulting in deafness, circling behavior, and dilute coat color in mice. The same mutation in any TRPML channel results in its functioning on the plasma membrane, where we showed they comprise proton-impermeant, Ca2+-permeant cation channels.
TRPV3 sensory channels. We identified a member of the human vanilloid TRP subfamily, TRPV3. TRPV3 is highly temperature sensitive and is found in skin cells, sensory neurons, and brain; it is sensitized with repeated heating and displays a marked hysteresis on heating and cooling. We have identified common plant compounds used as spices and insecticides that activate TRPV3, TRPV1, and other TRP channels.
Sperm are cells with crucial responsibilities; they must swim long distances to fuse with the egg and deliver the paternal DNA they carry. Although ATP-driven motors govern normal motility, ion channels initiate their turbocharged final burst, called hyperactivated motility. We identified four distinct genes (CatSpers1–4) that encode subunits of a sperm-specific calcium ion channel. By inventing a method to make intracellular recordings from sperm, we showed that the four CatSper proteins join to form a single Ca2+-selective pore. CatSpers are only present in the sperm tail; disruption of any CatSper gene abrogates hyperactivated motility and results in complete male sterility. We have purified the channel complex and shown that it contains three accessory subunits, whose functions we are investigating. These CatSper channels are novel targets for contraceptive drugs. We also identified a potassium current in sperm, KSper, whose properties are consistent with the mSlo3 K+ channel. We showed that alkalization of the spermatozoa is the trigger for activation of CatSpers and KSpers, which regulate Ca2+ entry and hyperactivated motility.
Mitochondrial Ca2+ Channels
Mitochondrial Ca2+ uptake controls the rate of energy production, shapes the amplitude and spatiotemporal patterns of intracellular Ca2+ signals, and is instrumental in cell death. This Ca2+ uptake is primarily via a 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; mitochondrial Ca2+ channel). This channel binds Ca2+ with extremely high affinity, enabling high Ca2+ selectivity despite relatively low cytoplasmic Ca2+ concentrations. MiCa is especially effective for Ca2+ uptake into energized mitochondria. We are attempting to identify the gene encoding this ion channel.
Hv1, the Voltage-Gated Proton Channel
We identified a novel, H+-selective, voltage-gated ion channel (Hv1). Hv1 currents rapidly alkalinize cells by extruding protons. Hv1 restores the imbalance of charge caused by NADPH oxidase electron transfer across the membrane, a mechanism used by organisms to kill bacteria and parasites. We deleted the Hv1 gene in mice and showed that Hv1 is required for effective oxidative burst activity in immune cells.
Hv1 also helps us understand voltage-gating mechanisms. Cation channels belong to a large class of proteins that function in cells, from bacteria to humans. Each channel subunit in a tetramer is formed by a polypeptide chain of six transmembrane-spanning segments (S1–S6). The self-contained voltage-sensing domain (VSD; S1–S4) is a positively charged "handle" that moves in response to transmembrane voltage changes. The VSD transmits force to the S5–S6 pore domain, thereby opening or closing the channel. Hv1 contains only a VSD domain, raising the question of how protons cross the membrane when the voltage sensor moves. The answer will yield important insights into channel gating movements.
Na+- and Ca2+-Selective Pores
We discovered a voltage-gated ion channel (NaChBac) from the extremophile bacteria Bacillus halodurans. Close relatives of this channel are Ca2+-selective. The channel expresses well in mammalian cells where its function can be examined. The identification of this simple channel will help us understand how more-complex mammalian voltage-gated cation-selective channels accomplish their functions. We are attempting 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+ or Ca2+ over other ions.
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