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Calcium-activated potassium channels modulate calcium signaling cascades and membrane potential in both excitable and non-excitable cells. In this article we will review the physiological properties, the structure activity relationships of the existing peptide and small molecule modulators and the therapeutic importance of the three small-conductance channels KCa2.1-KCa2.3 (a.k.a. SK1-SK3) and the intermediate-conductance channel KCa3.1 (a.k.a. IKCa1). The apamin-sensitive KCa2 channels contribute to the medium afterhyperpolarization and are crucial regulators of neuronal excitability. Based on behavioral studies with apamin and on observations made in several transgenic mouse models, KCa2 channels have been proposed as targets for the treatment of ataxia, epilepsy, memory disorders and possibly schizophrenia and Parkinson's disease. In contrast, KCa3.1 channels are found in lymphocytes, erythrocytes, fibroblasts, proliferating vascular smooth muscle cells, vascular endothelium and intestinal and airway epithelia and are therefore regarded as targets for various diseases involving these tissues. Since two classes of potent and selective small molecule KCa3.1 blocker, triarylmethanes and cyclohexadienes, have been identified, several of these postulates have already been validated in animal models. The triarylmethane ICA-17043 is currently in phase III clinical trials for sickle cell anemia while another triarylmethane, TRAM-34, has been shown to prevent vascular restenosis in rats and experimental autoimmune encephalomyelitis in mice. Experiments showing that a cyclohexadiene KCa3.1 blocker reduces infarct volume in a rat subdural hematoma model further suggest KCa3.1 as a target for the treatment of traumatic and possibly ischemic brain injury. Taken together KCa2 and KCa3.1 channels constitute attractive new targets for several diseases that currently have no effective therapies.

In insects, increasing evidence suggests that small secreted pheromone binding proteins (PBPs) and odorant binding proteins (OBPs) are important for normal olfactory detection of airborne pheromones and odorants far from their source. In contrast, it is unknown whether extracellular ligand binding proteins participate in perception of less volatile chemicals, including many pheromones, that are detected by direct contact with chemosensory organs. CheB42a, a small Drosophila melanogaster protein unrelated to known PBPs or OBPs, is expressed and likely secreted in only a small subset of gustatory sensilla on males' front legs, the site of gustatory perception of contact pheromones. Here we show that CheB42a is expressed specifically in the sheath cells surrounding the taste neurons expressing Gr68a, a putative gustatory pheromone receptor for female cuticular hydrocarbons that stimulate male courtship. Surprisingly, however, CheB42a mutant males attempt to copulate with females earlier and more frequently than control males. Furthermore, CheB42a mutant males also attempt to copulate more frequently with other males that secrete female-specific cuticular hydrocarbon pheromones, but not with females lacking cuticular hydrocarbons. Together, these data indicate that CheB42a is required for a normal gustatory response to female cuticular hydrocarbon pheromones that modulate male courtship.

Small conductance Ca 2؉ -activated K ؉ channels, products of the SK1-SK3 genes, regulate membrane excitability both within and outside the nervous system. We report the characterization of a SK3 variant (SK3-1C) that differs from SK3 by utilizing an alternative first exon (exon 1C) in place of exon 1A used by SK3, but is otherwise identical to SK3. Quantitative RT-PCR detected abundant expression of SK3-1C transcripts in human lymphoid tissues, skeletal muscle, trachea, and salivary gland but not the nervous system. SK3-1C did not produce functional channels when expressed alone in mammalian cells, but suppressed SK1, SK2, SK3, and IKCa1 channels, but not BK Ca or K V channels. Confocal microscopy revealed that SK3-1C sequestered SK3 protein intracellularly. Dominant-inhibitory activity of SK3-1C was not due to a nonspecific calmodulin sponge effect since overexpression of calmodulin did not reverse SK3-1C-mediated intracellular trapping of SK3 protein, and calmodulin-Ca 2؉ -dependent inactivation of Ca V channels was not affected by SK3-1C overexpression. Deletion analysis identified a dominant-inhibitory segment in the SK3-1C C terminus that resembles tetramerization-coiled-coiled domains reported to enhance tetramer stability and selectivity of multimerization of many K ؉ channels. SK3-1C may therefore suppress calmodulin-gated SK Ca /IK Ca channels by trapping these channel proteins intracellularly via subunit interactions mediated by the dominant-inhibitory segment and thereby reduce functional channel expression on the cell surface. Such family-wide dominant-negative suppression by SK3-1C provides a powerful mechanism to titrate membrane excitability and is a useful approach to define the functional in vivo role of these channels in diverse tissues by their targeted silencing.

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types. This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells. Protocol DiscussionGreat advances in our understanding of CNS development and stem cell biology have been made possible by our ability to harvest, isolate and culture embryonic neural stem cells. This video demonstrates the dissection of E12.5 mouse cerebral cortex and the subsequent disaggregation and culturing of embryonic neural stem cells. Many other other similar methods have been successfully employed by other investigators. 2. Skin and mesenchymal layers were removed from dissected telencephalic vesicles. 3. Vesicles were incubated in 0.05% trypsin with 0.02% EDTA and 0.2% BSA in HBSS for 20 minutes at 37°C. 4. Trypsinization was stopped by an equal volume of 1 mg/ml soybean trypsin inhibitor (Sigma #T6522) in HBSS. 5. Tissue digests were dissociated using several rounds of trituration with fire-polished Pasteur pipettes. 6. Cells were washed once with 0.2% BSA in HBSS and plated at 50,000 cells/ml on laminin-coated coverslips in media with 20 ng/ml EGF, 10 ng/ml FGF2 (R&D Systems or Peprotech), and 2 ug/ml heparin (Sigma).

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting. This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex. The digestion of the medulla into single chromaffin cells is then demonstrated.

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Historically, JoVE, The Journal of Visualized Experiments, has focused primarily on biomedical research and has developed subsections for Bioengineering, Clinical and Translational Medicine, Immunology and Infection, and Neuroscience. This July, JoVE launches its Applied Physics section, which includes a range of content from Plasma Physics to Materials Science. We begin the new section with a notable article from Purdue University, where researchers in the Center for Laser-Based Manufacturing are studying.Matter exists in three familiar states: solid, liquid, and gas. If a gas is hit with enough energy, atoms can lose electrons, or become ionized, to form a fourth state called plasma. Plasma is the most abundant form of matter; occupying about 99.999% of the visible universe. Using ultrashort laser pulses of 100 femtoseconds, or 100 quadrillionths of a second, our authors demonstrate a technique called pump probe shadowgraphy, which allows the early plasma to be visualized as it evolves from metal surfaces. By constructing a simulation model, these investigators are able to examine early plasma dynamics, enabling a better understanding of how matter becomes ionized.For the materials science subcategory of applied physics, JoVE materializes at the University of Michigan, where researchers are developing new methods in microfabrication. Our authors demonstrate a method for growing complex, three-dimensional microstructures out of carbon nanotubes, which can be used as master molds to cast replicas out of polymers or biological materials. Scanning electron microscopy reveals that the carbon nanotube master molds are reproduced with high fidelity in microscale shape and nanoscale texture in the polymer replicas made from these molds. Microfabrication technology allows laboratory operations to be performed on small scales -essentially putting a lab on a chip.Shifting from Applied Physics to cardiac physiology, JoVE visits The George Washington University to capture a modified Langendorff preparation. JoVE has published several articles that demonstrate this physiological prep, which allows the heart to beat in isolation for hours at a time. The approach involves retrograde perfusion of the heart, via the aorta, which shuts the aortic valve and forces oxygenated perfusate through the coronary circulation, in order to sustain cardiac tissue.Our authors present a modification to this preparation that involves cannulating the left atrial appendage, the inferior vena cava, and the pulmonary artery. In this state all four chambers of the heart are cannulated, thereby providing physiological load pressures to both ventricles, and eliminating the need to retrogradely-pefuse the heart through the aorta. Therefore, perfusion of the heart is in the normal direction, and the heart provides its own pressure for coronary perfusion.Once this biventricular working heart model is achieved, our authors proceed to set up imaging of nictonamide adenine dinucleoutide, or NADH, fluorescence. This coenzyme, which is found in mitoc...