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 300 genes encoding ion channels, the cell's transistors. My laboratory finds and investigates ion channels that regulate Ca2+ signaling, both across the plasma membrane and across the membranes of organelles such as mitochondria.
Na+- and Ca2+-Selective Pores
Voltage-gated sodium (Nav) channels are essential for the rapid depolarization of nerve and muscle and are important drug targets. We discovered a voltage-gated ion channel (NaChBac) from the extremophile bacteria Bacillus halodurans. The channel expresses well in mammalian cells, where its function can be examined. In collaboration with Nieng Yan (Tsinghua University) and colleagues, we expressed and characterized NavRh, a NaChBac ortholog from the marine alphaproteobacterium Rickettsiales (denoted Rh), and determined its structure to 3.05-Ǻ resolution (Figure 1). The carbonyl oxygen atoms of Thr178 and Leu179 constitute a hydrated calcium ion–binding site within the selectivity filter. The outer mouth of the Na+ selectivity filter, defined by Ser181 and Glu183, is closed, as is the activation gate at the intracellular side of the pore. The voltage sensors adopt a depolarized conformation in which all the gating charges are exposed to the extracellular environment. Close relatives of these bacterial voltage-gated Na+ channels primarily conduct calcium ions, and these are candidates for the high-resolution structure of Ca2+-selective channel pores. Most recently, we have mutagenized NaChBac homologs and, in collaboration with Bonnie Wallace (University College London), examined these currents and crystal structures to understand sodium selectivity and blocking by NaV channel blockers.
Hv1, the Voltage-Gated Proton Channel
We (and, independently, the Yasushi Okamura laboratory, Okazaki Institute) identified a novel, H+-selective, voltage-gated ion channel (Hv1). Unlike ion channels that conduct other cations, Hv1 contains only a voltage sensor domain. Voltage changes uncover a pathway for protons to navigate across the protein from inside to outside the cell. Hv1 currents rapidly alkalinize cells by extruding protons. Hv1 restores the imbalance of charge caused by NADPH oxidase (NOX) 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. We found that mouse and human brain microglia, but not neurons or astrocytes, express large Hv1-mediated currents. Hv1 is required for NOX-dependent ROS generation in brain microglia in situ and in vivo. Mice lacking Hv1 are protected from NOX-mediated neuronal death and brain damage 24 hours after stroke. These results demonstrate that Hv1-dependent ROS production is responsible for a substantial fraction of brain damage at early time points after ischemic stroke.
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. Trpm7 is found in practically all cells of vertebrates and its deletion is lethal at an early embryonic stage. 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 ion channel gene is one of few to have such a fundamental role in developmental processes, with implications for all aspects of vertebrate biology. We are focusing on obtaining the high-resolution structure of areas within this large channel/kinase and on understanding the kinase substrates.
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. When any of the TRPC1, C4, or C5 genes is deleted in mice, the 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. We are investigating TRPC1, C4, and C5 function in brains of mice in which any combination of these three genes have been deleted.
Spermatozoa-Specific Ion Channels
Spermatozoa 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 (Figure 2), 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 at least four accessory subunits, whose functions we are investigating. We also identified a potassium current in sperm, KSper, which we and others identified as 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.
Mammalian spermatozoa undergo complex adaptations within the female (the process of capacitation) that are initiated by agents ranging from pH to progesterone, but these factors are not necessarily taxic. Currently, hemotaxis, thermotaxis, and rheotaxis have not been definitively established in mammals. We showed that positive rheotaxis, the ability of organisms to orient and swim against the flow of surrounding fluid, is a major taxic factor for mouse and human sperm. This flow is generated in female mice within 4 hours of sexual stimulation and coitus; prolactin-triggered oviductal fluid secretion clears the oviduct of debris, lowers viscosity, and generates the stream that guides sperm migration in the oviduct. Rheotaxic movement was demonstrated in capacitated and uncapacitated spermatozoa in low- and high-viscosity media. Finally, we showed that a unique sperm motion, which we quantify using the sperm head's rolling rate, reflects sperm rotation that generates essential force for positioning the sperm in the stream. Rotation requires CatSper channels that enable Ca2+ influx.
We used genetics, superresolution fluorescence microscopy, and phosphoproteomics to investigate the CatSper-dependent mechanisms underlying this flagellar switch. We found that the CatSper channel is required for four linear calcium domains that organize signaling proteins along the flagella. This unique structure focuses tyrosine phosphorylation in time and space as sperm acquire the capacity to fertilize. In heterogeneous sperm populations, we found unique molecular phenotypes, but only sperm with intact CatSper domains that organize time-dependent and spatially specific protein tyrosine phosphorylation successfully migrate. These findings illuminate flagellar adaptation, signal transduction cascade organization, and fertility.
Over the last 10 years we have identified ion channels whose function is primarily on organellar membranes (Figure 3).
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 (Figure 4), 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 working with Vamsi Mootha’s lab (HHMI, Massachusetts General Hospital) to determine the components of the uniporter and their affects on its function. We also identified Letm1, a gene mutant in Wolf-Hirschhorn syndrome, as being a mitochondrial Ca2+/H+ exchanger in the inner mitochondrial membrane.
Ion Channels in Cilia. Cilia are protrusions from the cell that are less than the width of a single mitochondrion, but that can be as long as a cell. One new area of our research is to determine the ion channels in cilia. We recently introduced the whole-cilium patch-clamp recording method and used it to identify ion channels specifically in cilia (Figure 5). We also developed the Arl13b-EGFP ciliary-marker and Arl13b-mCherry-GECO1.2 calcium-sensor transgenic mice to report [Ca2+] in live cilia. These approaches and new methods will address an important bottleneck in understanding how cilia sense their surroundings and respond to control cell growth and cell division. These innovations will help the field evolve from its strong genetically defined understanding of ciliopathies to a more mechanistic-based understanding.
Two-Pore Sodium Channels in Endolysosomes. Mammalian TPC1 and TPC2 genes encode two-pore ion channels that are localized to intracellular endosomes and lysosomes. By directly recording TPCs under voltage clamp in endolysosomes from wild-type and TPC double-knockout mice, we and the laboratories of Haoxing Xu (University of Michigan) and Dejian Ren (University of Pennsylvania) showed that TPCs are sodium-selective channels activated by PI(3,5)P2. These findings may explain the specificity of PI(3,5)P2 in regulating the fusogenic potential of intracellular organelles. Furthermore, we showed that the primary endolysosomal ion is Na+, not K+, as had been previously assumed. In further studies, we and the Ren laboratory showed that TPCs are ATP sensitive. The channel complex detects nutrient status, becomes constitutively open upon nutrient removal and mTOR translocation from the lysosomal membrane, and controls the lysosome's membrane potential, pH stability, and amino acid homeostasis. Mutant mice lacking lysoNaATP have reduced exercise endurance after fasting. Thus, TPCs make up an ion channel family that couples the cell's metabolic state to endolysosomal function, and they are important for physical endurance during food restriction.
Grants from the National Institutes of Health, the Bill and Melinda Gates Foundation, and the Manton Foundation provided partial support for this work.
As of February 14, 2014