2Selective Permeability – What is it? Selective permeability means that the cell membrane has some control over what can cross it, so that only certain molecules either enter or leave the cell.
3Regulation of Membrane Permeability Ionic composition inside neurons is tightly regulated and must be able to be rapidly modified in response to various stimuliSpecialized transmembrane channels allow ions to rapidly enter the cell while still maintaining high selelctivityVoltage-gated channelsLigand-gated channelsMechano-sensitive channels
4Neural SignalingResting potential is the result of an equilibrium between the electrical and chemical forces acting upon Na+ and K+Depolarization is result of a stimulus large enough to cause voltage-gated Na+ channels to openRepolarization is the closing of Na+ channels, increasing the relative permeability to K+
5What properties govern the specificity of channels What properties govern the specificity of channels? How can this be studied? How can we understand this in terms of dysfunction and disease?
6Techniques X-ray crystallography Electron Density Maps Pharmacology GeneticsSequencingSite-directed mutagenesisPatch-clamping
7Voltage-Gated Ion Channels Voltage-gated ion channels are like the transistors of logical circuits, detecting, amplifying, and reshaping electrical messagesHodgkin and Huxley succeeded in describing the permeability changes as a set of chemical reactions whose rate constants are a function of voltage“…changes in ionic permeability depend on the movement of some component of the membrane which behaves as though it had a large charge or dipole movement…to allow ions to pass when they occupy particular sites in the membrane.”Predicted the “gating current” which is a small charge movement generated by the voltage-driven conformational changes that open the channels
8Visualization of the channel Structure and function has been studied using X-ray crystallographic studiesElucidated an important feature of the selectivity filter to create a queue of cation binding sites that mimic the waters of hydration surrounding that specific cationAllows the observation of the channel structure in various chemical environmentsAllowed the visualization of ion position within the filterReduction of K+ to 3mM in the presence of 150mM Na+ causes the selectivity filter to undergo a conformational change to a collapsed state
9General Features of a VGIC Armstrong et al. 1998SF = Selectivity FilterVS = Voltage SensorOV = Outer VestibuleIV = Inner VestibuleVoltage Gated: Pore opening is energetically coupled to the movement of a charged voltage sensor within the membrane electric fieldConformational changes underlying the voltage sensing property are large and involve movements of arginine residues through the membrane near the protein-lipid interface
10Voltage Sensor (S4 Segment) Cysteine mutagenesis and cysteine labeling with thiol-reactive compounds has provided good evidence that the S4 segments in both Na+ and K+ channels move as expected following voltage changesState dependent labeling a specific cysteine may be labeled only from the outside when the channel is open and only from the inside when the channel was closed. The labeling patterns are consistent with the outward movement of S4 (the voltage sensor) in response to a positive change of membrane potential
11Potassium ChannelPotassium conduction through their specific channels is critical for cell volume regulation, hormone secretion, and electrical impulse formation (neural signaling)Signature Sequence forms the selectivity filter which prevents the passage of sodium ions but allows potassium ions to conduct across the membrane at rates approaching the diffusion limit.MacKinnon. 2003
12Channel ArchitectureSince the inner helices are tilted slightly inward and kinked with respect to the membrane, the subunits open like the petals of a flower facing the outside of the cell. Gives the appearance of an inverted teepeeThis architecture is likely a general feature of cation channels with four inner helices, four pore helices, and a selectivity filter each tuned to select the specific ion at the extracellular surfaceMacKinnon et al. 1998
13The channel pore is made up of four subunits, each of which contain two fully transmembrane α-helices (an inner and outer) and a tilted pore helix that runs half-way through the membrane and points its negative end charged C-terminus toward the ion pathwayMacKinnon et al. 1998
14Ion Selectivity and Movement A K+ ion could move through the internal pore and cavity without shedding its waters of hydration but the selectivity pore is so narrow that it must shed its hydrating waters to enter. The ion is stabilized in the selectivity pore because it is lined exclusively with polar main chain atoms found in the signature sequence.The configuration of these ions within the selectivity pore allows the channel to exploit the electrostatic repulsive forces to overcome the attractive forces between K+ ions and the selectivity filter
15As the ion encounters the selectivity filter, it interacts with 4 evenly spaced layers of carbonyl oxygen atoms and a single layer of threonine hydroxyl oxygen atoms this creates 4 binding sites where K+ binds in a dehydrated state and is surrounded by 8 oxygens that essentially mimic the arrangement of water molecules around a hydrated ion that is observed in the central cavityMacKinnon. 2003
16Visualization of Ion Position All K+ channels show a selectivity sequence of K+ ≈ Rb+ > Cs+. Permeability for the smaller ions such as Na+ and Li+ are immeasurably lowRb+ (1.48Å) is almost a perfect analog to K+ (1.33Å) due to the similarity in size and permeability characteristics. Rb+ (as well as Cs+) is more electron dense than K+ making it very suitable for use in visualizing the locations of permeant ions in the pore using electron density mapsMacKinnon et al. 1998
17Isolating the ChannelAll K+ channels can be blocked by tetraethylammonium (TEA)TEA interacts with the amino acids that define the entryway into the pore and blocks the K+ channel at both sides of the membrane at distinct sites. The amino acids that TEA cations interact with are located just external to and internal to the structure that forms the selectivity filter. TEA cannot enter the selectivity filter, only block it.TEA ions can only traverse ~20% of the transmembrane voltage difference which suggests that ~80% of the transmembrane voltage is imposed across the relatively short distance through the selectivity filter which is ~12Å (Length of pore = 45Å) the rate-limiting steps for a K+ to travel through the channel occur in the short span of the selectivity filter
18Functional Experiments The transmembrane voltage is imposed across the relatively short distance through the selectivity filter which is ~12Å (Length of pore = 45Å) the rate-limiting steps for a K+ to travel through the channel occur in the short span of the selectivity filterTEA ions can only traverse ~20% of the transmembrane voltage
19Summary of K+ ChannelThe pore is constructed like an inverted teepee, with the selectivity filter held at its wide end. This is most likely applicable to other cation channels such as Ca2+ and Na+The narrow selectivity filter is ~12Å long and has a polar lining, whereas the remainder of the pore is wider and has a relatively inert hydrophobic lining. This favors high K+ throughput by minimizing the distance over which K+ interacts strongly with the channelThe large water filled cavity and helix dipoles help to overcome the high electrostatic energy barrier in the low dielectric membrane centerThe K+ selectivity filter is lined with carbonyl oxygen atoms which provide 4 closely spaced binding sites and is constrained to an optimal geometry that will only fit a dehydrated K+ ionTwo K+ ions in close proximity within the filter exude repulsive forces on each other, which helps to overcome the otherwise strong interaction between ion and protein. This further allows for rapid conduction while still favoring high selectivity.
21InactivationInactivation gating is a second gating factor that is mechanistically simpler and different from activation after the activation gate opens, a portion of the channel peptide diffuses into the mouth of the inner vestibule of the pore and blocks conduction (a foot in the door sort of mechanism)
22Inactivation (cont.)Site-directed mutagenesis by Aldrich et al. found that deletions within the first 20 amino acids of the N-terminus completely destroy fast inactivation. Even more fascinating was the finding that deletions in the next 63 amino acids resulted in speeding inactivationCutting off the inactivation particle with pronase removed the inactivation ability
23CNS PathologiesMany of the ion channel diseases are known as paroxysmal disorders in which the principal symptoms occur intermittently in some individuals who otherwise may be healthy and active
24EpilepsyEpileptic seizures are behavioral attacks resulting from the overly synchronized and excessive activity of large groups of neurons.Symptoms: Alterations/loss of consciousness, sustained or rhythmic muscle contraction, stereotyped gestural movements, and visual/somatosensory hallucinationsAltered Na+ channel function has been linked to epilepsy
25Epilepsy (cont.)The SCN1B (the β1 auxiliary subunit of the voltage gated Na+ channel) genes of epileptic individuals contained a single base-pair substitution which caused a single amino acid change in the protein sequenceThe β1 subunit is important for the timing of channel open and closeA mutation in this region results in a loss of function cells expressing the mutant exhibit slow channel openings and closings
26On a larger scale…The blood-brain barrier (BBB) provides a stable environment for neural function through a combination of specific ion channels and transporters which keep the ionic composition optimal for synaptic signaling.Functions as a protective barrier that shields the CNS from neurotoxic substances that is present in the blood.Dysfunction of the BBB has been found in several CNS pathologies such as stroke, trauma, infectious or inflammatory processes, MS, HIV, Alzheimer’s, Parkinson’s, epilepsy, brain tumors, pain, and glaucoma
27There are 3 main barrier sites between the blood and the brain: The BBB is created by the endothelial cells that form the walls of capillaries.There are 3 main barrier sites between the blood and the brain:The BBB proper – created at the level of the cerebral capillary endothelial cells by tight junction formation. No brain cell is further than ~25nm from a capillary so diffusion distances are shortThe blood-CSF barrierThe arachnoid barrier – the brain is enveloped by the arachnoid membrane lying under the dura
28At all three of the barriers, the function is a result of a physical barrier formed by tight junctions, a transport barrier formed by specific transport mechanisms mediating solute flux, and a metabolic barrier formed by enzymes metabolizing molecules in transit).Abbot et al. 2009
29Tight JunctionsTight junctions act as a fence in the membrane and segregate transport proteins and lipid rafts, to either the luminal or abluminal membrane domain, and to prevent their free movement from one side of the epithelium to the other in order to preserve the polarity of the barrier.Abbot et al. 2009
30Selective TransportSpecific transport mechanisms must exist to supply the brain with just the right amount of essential water soluble nutrients and metabolites that are required by nervous tissueAbbot et al. 2009
31Thank You!!!References:Abbott, N.J., et al., Structure and function of the blood–brain barrier. Neurobiol. Dis. (2009).Armstrong, CM and Hille B. Voltage-gated ion channels and electrical excitability. Neuron Vol. 20, pp. 371–380 (1998).Cooper EC, Yeh Jan L. Ion Channel Genes and Human Neurological Disease: Recent Progress, Prospects, and Challenges. PNAS, Vol. 96, pp (1999).MacKinnon R. Minireview: Potassium Channels. FEBS Letters 555, pp (2003).Mackinnon R., et al. The Structure of the Potassium Channel: Molecular Basis of K+ Conduction and Selectivity. Science Vol. 280, pp (1998).