Project description:Genomic profile of transporters and ion channels in differentiated or undifferentiated Caco-2 cells grown for 5 days, 1 week, 2 weeks or 3 weeks in flasks or filters Keywords: ordered
Project description:Specific macromolecular transport systems, ion channels and pumps, provide the pathways to facilitate and control the passage of ions across the lipid membrane. Ion channels provide energetically favourable passage for ions to diffuse rapidly and passively according to their electrochemical potential. Selective ion channels are essential for the excitability of biological membranes: the action potential is a transient phenomenon that reflects the rapid opening and closing of voltage-dependent Na+-selective and K+-selective channels. One of the most critical functional aspects of K+ channels is their ability to remain highly selective for K+ over Na+ while allowing high-throughput ion conduction at a rate close to the diffusion limit. Permeation through the K+ channel selectivity filter is believed to proceed as a 'knockon' mechanism, in which 2-3 K+ ions interspersed by water molecules move in a single file. Permeation through the comparatively wider and less selective Na+ channels also proceeds via a loosely coupled knockon mechanism, although the ions do not need to be fully dehydrated. While simple structural concepts are often invoked to rationalize the mechanism of ion selectivity, a deeper analysis shows that subtle effects play an important role in these flexible dynamical structures.
Project description:The study of ion channels dates back to the 1950s and the groundbreaking electrophysiology work of Hodgin and Huxley, who used giant squid axons to probe how action potentials in neurons were initiated and propagated. More recently, several experiments using different structural biology techniques and approaches have been conducted to try to understand how potassium ions permeate through the selectivity filter of potassium ion channels. Two mechanisms of permeation have been proposed, and each of the two mechanisms is supported by different experiments. The key structural biology experiments conducted so far to try to understand how ion permeation takes place in potassium ion channels are reviewed and discussed. Protein crystallography has made, and continues to make, key contributions in this field, often through the use of anomalous scattering. Other structural biology techniques used to study the contents of the selectivity filter include solid-state nuclear magnetic resonance and two-dimensional infrared spectroscopy, both of which make clever use of isotopic labeling techniques, while molecular-dynamics simulations of ion flow through the selectivity filter have been enabled by the growing number of potassium ion channel structures deposited in the Protein Data Bank.
Project description:We describe the incorporation of gated ion channels into probes for scanning ion conductance microscopy (SICM) as a robust platform for collecting spatial information at interfaces. Specifically, a dual-barrel pipet is used, where one barrel controls the pipet position and the second barrel houses voltage-gated transient receptor potential vanilloid 1 (TRPV1) channels excised in a sniffer-patch configuration. Spatially resolved sensing with TRPV1 channels is demonstrated by imaging a porous membrane where a transmembrane potential across the membrane generates local electric field gradients at pores that activate TRPV1 channels when the probe is in the vicinity of the pore. The scanning routine and automated signal analysis demonstrated provide a generalizable approach to employing gated ion channels as sensors for imaging applications.
Project description:Phosphoinositides serve as signature motifs for different cellular membranes and often are required for the function of membrane proteins. Here, we summarize clear evidence supporting the concept that many ion channels are regulated by membrane phosphoinositides. We describe tools used to test their dependence on phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate, and consider mechanisms and biological meanings of phosphoinositide regulation of ion channels. This lipid regulation can underlie changes of channel activity and electrical excitability in response to receptors. Since different intracellular membranes have different lipid compositions, the activity of ion channels still in transit towards their final destination membrane may be suppressed until they reach an optimal lipid environment. This article is part of a Special Issue entitled Phosphoinositides.
Project description:Migraine is one of the most common neurological disorders. Despite its prevalence, the basic physiology of the molecules and mechanisms that contribute to migraine headache is still poorly understood, making the discovery of more effective treatments extremely difficult. The consistent presence of head-specific pain during migraine suggests an important role for activation of the peripheral nociceptors localized to the head. Accordingly, this review will cover the current understanding of the biological mechanisms leading to episodic activation and sensitization of the trigeminovascular pain pathway, focusing on recent advances regarding activation and modulation of ion channels.
Project description:In Alzheimer's disease, calcium permeability through cellular membranes appears to underlie neuronal cell death. It is increasingly accepted that calcium permeability involves toxic ion channels. We modeled Alzheimer's disease ion channels of different sizes (12-mer to 36-mer) in the lipid bilayer using molecular dynamics simulations. Our Abeta channels consist of the solid-state NMR-based U-shaped beta-strand-turn-beta-strand motif. In the simulations we obtain ion-permeable channels whose subunit morphologies and shapes are consistent with electron microscopy/atomic force microscopy. In agreement with imaged channels, the simulations indicate that beta-sheet channels break into loosely associated mobile beta-sheet subunits. The preferred channel sizes (16- to 24-mer) are compatible with electron microscopy/atomic force microscopy-derived dimensions. Mobile subunits were also observed for beta-sheet channels formed by cytolytic PG-1 beta-hairpins. The emerging picture from our large-scale simulations is that toxic ion channels formed by beta-sheets spontaneously break into loosely interacting dynamic units that associate and dissociate leading to toxic ionic flux. This sharply contrasts intact conventional gated ion channels that consist of tightly interacting alpha-helices that robustly prevent ion leakage, rather than hydrogen-bonded beta-strands. The simulations suggest why conventional gated channels evolved to consist of interacting alpha-helices rather than hydrogen-bonded beta-strands that tend to break in fluidic bilayers. Nature designs folded channels but not misfolded toxic channels.
Project description:Biological ion channels are nanoscale transmembrane pores. When water and ions are enclosed within the narrow confines of a sub-nanometer hydrophobic pore, they exhibit behavior not evident from macroscopic descriptions. At this nanoscopic level, the unfavorable interaction between the lining of a hydrophobic pore and water may lead to stochastic liquid-vapor transitions. These transient vapor states are "dewetted", i.e. effectively devoid of water molecules within all or part of the pore, thus leading to an energetic barrier to ion conduction. This process, termed "hydrophobic gating", was first observed in molecular dynamics simulations of model nanopores, where the principles underlying hydrophobic gating (i.e., changes in diameter, polarity, or transmembrane voltage) have now been extensively validated. Computational, structural, and functional studies now indicate that biological ion channels may also exploit hydrophobic gating to regulate ion flow within their pores. Here we review the evidence for this process and propose that this unusual behavior of water represents an increasingly important element in understanding the relationship between ion channel structure and function.
Project description:This paper looks at ion channels as an example of the pharmacologist's stock in trade, the action of an agonist on a receptor to produce a response. Looked at in this way, ion channels have been helpful because they are still the only system which is simple enough for quantitative investigation of transduction mechanisms. A short history is given of attempts to elucidate what happens between the time when agonist first binds, and the time when the channel opens.