readerL3 - Channel Structure The activation or deactivation...

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Unformatted text preview: Channel Structure The activation or deactivation of ion channels is responsible for all electrical signaling in the nervous system. In order to understand the function of ion channels, it is very helpful to know the structure. nAChR: nicotinic acetylcholine receptor Historically, the best studied ion channel is the nicotinic acetylcholine receptor (nAChR). The nAChR is found on the postsynaptic membrane of neurons in various areas of vertebrate and invertebrate nervous systems, on muscle fibers at the vertebrate neuromuscular junction (NMJ), and in very high concentration in the electric organ of the electric fish (Torpedo rays). (1) The nAChR is called nicotinic because nicotine is an agonist (that is a substance that mimics the effects of the endogenous neurotransmitter). Page 3-1 (2) The nAChR is an ionotropic neurotransmitter receptor. This means that the receptor is itself an ion channel, and when the neurotransmitter ACh binds to the receptor and activates it, the pore in the channel opens allowing cations to flow through. (3) Another kind of AChR, the muscarinic AChR is a metabotropic receptor. This means that the receptor is not a channel, but rather that neurotransmitter binding to the receptor activates a second messenger cascade within the neuron that eventually results in the opening or closing of ion channels. The structure of nAChR: (1) The nAChR from electric organ or muscle consists of four different subunits, , , and . The nAChR from neurons has only and subunits. (2) In a functional nAChR there are five subunits, two , and three non- . These subunits form a cylinder around a central pore. ACh binds to the subunits. (3) The cDNA for each subunit has been cloned, and the amino acid sequences have been determined. The four different subunits are highly homologous and therefore have very similar structures. Page 3-2 Determining the structure of AChR A number of techniques can be used to determine the structure of a protein. (1) Hydrophobicity plots: We can calculate hydrophobicity from the amino acid sequence in order to identify hydrophobic membrane spanning regions of the molecule. Based on hydrophobicity plots, each nAChR subunit has four membrane spanning regions. The amino terminus of the molecule is extracellular. (A) It is preceded by a signal sequence, indicating that it is secreted from the cell. (B) Two amino acids at the amino(N)-terminus of the molecule are known to be associated with Ach binding to the subunit. (C) The portion of the molecule from the amino terminus to the beginning of the first membrane spanning region represents about 1/2 of the molecule, agreeing with electron microscope pictures of the molecule. (D) Since there are an even number of membrane spanning regions (four) the carboxy(C)-terminus must also be extracellular. Page 3-3 (2) Site-directed mutagenesis : In order to determine which amino acids line the pore of the nAChR molecule, site-directed mutagenesis has been used. This technique can alter particular amino acid residues in the molecule. The mutated molecule can then be expressed in Xenopus eggs, where the effects of the mutation on channel function can be determined. If particular amino acids do line the pore, then mutations in those amino acids would be expected to alter the receptors conductance or ion specificity. (3) High-resolution imaging : High-resolution electron microscope imaging resolve the channel topology to better than nm range, thereby revealing many details of the structure. Predicted nAChR structure Page 3-4 Of particular interest in this structure is the kink in membrane spanning region number 2 (M2). This kink is what closes the pore. The M2 regions rotate outwards when ACh binds, forming an open pore. A receptor superfamily: A number of other ionotropic neurotransmitter receptors have also been cloned. The close similarity of amino acid composition of their subunits suggests that these receptor have a common genetic origin, forming a receptor superfamily. (1) Shown in the right are the hydrophobicity plots for the nAChR and two types of GABA ( aminobutyric acid) receptors. The three subunits have very similar profiles. Glycine and serotonin receptors also appear to have similar subunit structures. All of these receptors consist of five subunits. (2) GABA and glycine receptors are selective for anions (in vivo they let through only chloride) while serotonin and nAChRs are selective for cations (letting through potassium and sodium). This is surprising given their high sequence homology and similar structures. One difference is that the cation-selective channels have many negatively charged amino acids lining the vestibule ( the extracellular part of the molecule) which would tend to concentrate cations and exclude anions, while anion-selective channels have positively charged vestibule linings, which would do the opposite. Page 3-5 Voltage-gated channels: Ion channels activated the binding of a ligand like a neurotransmitter (Ach, GABA, glycine, etc.) are classified as ligand-gated channels. A second major class of channels are voltage-gated channels. Voltage-gated channels are activated by changes in membrane potential: (1) Voltage gated sodium and potassium channels underlie the generation of action potentials. (2) Voltage gated calcium channels are important for neurotransmitter release. Page 3-6 Structure The structures of the voltage gated channels have been determined using the same techniques that were used for finding the structure of the nAChR. nAChR (1) The voltage-gated sodium and voltage-gated calcium channels each consist of one large molecule with four similar domains. Each domain has six membrane spanning regions. Membrane spanning region S4 has every third amino acid being a positively charged arginine or lysine. This charged region is thought to cause the molecules' activation in response to changes in membrane potential. Voltage-gated sodium channel Voltage-gated calcium channel Page 3-7 (2) The voltage-gated potassium channel is made up of four subunits, with each subunit resembling a domain in the voltage gated sodium or calcium channel. (3) The loop between S5 and S6 form the lining of the extracellular portion of the pore and give the channel its ion selectivity. Mutations in this region can cause a sodium channel to act much like a calcium channel, no longer being selective for sodium. (2) The exact mechanisms that give channels ion specificity are not known, however pore size is very important: Sodium channels have a pore size too small to allow potassium to enter. Potassium channels, on the other hand, have a pore size that just fits a dehydrated potassium ion: Lining the entrance to the pore are rings of four carbonyl oxygens (one from each subunit) strip the water molecules from potassium ions allowing them to enter. Hydrated sodium ions are too large to enter the pore, and cannot be dehydrated by the carbonyl oxygens because they are too small to interact with all four carbonyl oxygens at the same time. Voltage-gated potassium channel Importance of active transport When an ion channel is activated, ions flow through it passively, along their electrochemical gradients. Over time, such ion fluxes would cause a breakdown of the electrochemical gradient such that eventually all ions would be found in equal concentrations inside and outside of the cell. If this occurred, neurons would no longer be able to generate electrical signals, and the nervous system could not function. Ion transporters are molecules that move ions across the membrane to maintain constant ionic concentrations inside the neuron. Since these ion movements are against the electrochemical gradient, the transporters require energy to work. (1) Primary active transport uses energy produced by hydrolyzing ATP. (2) Secondary active transport uses the energy of sodium moving down its electrochemical gradient to transport other ions across the membrane. Sodium potassium ATPase (1) The sodium potassium ATPase (or sodium potassium exchange pump) transports sodium out of the cell and potassium back into the cell. (2) For every molecule of ATP hydrolyzed, three sodium ions are transported out and two potassium ions are transported in. Because of the unequal number of ions being transported into and out of the cell, the sodium potassium ATPase causes a small change in membrane potential. (2) The pump cause a hyperpolarization. This makes a very small contribution to the resting membrane potential of the cell. Transporter Page 3-8 Page 3-9 Calcium ATPases Calcium acts as a signaling molecule within a neuron. Transient increases in calcium concentration are needed to release neurotransmitter at a presynaptic terminal, and as a second messenger in a number of processes. In order for calcium signaling to work, intracellular calcium levels must be kept low. Calcium ATPases in the cell membrane and in the membranes of intracellular organelles pump calcium out of the cytoplasm. Sodium calcium exchanger (1) Sodium calcium exchanger (NCX transport system) moves one calcium molecule out of the cell against its electrochemical gradient for every three sodium molecules that it allows to move into the cell down their electrochemical gradient. (2) This type of transport uses the energy stored in the sodium gradient, energy that must be replenished by the sodium potassium ATPase. (3) The exchange is not electrically neutral: each forward cycle transfers one positive charge into the cell. (4) The sodium calcium exchanger can run backwards (making sodium leave the cell and calcium enter the cell) under certain physiological conditions or by altering one or more of the ionic gradients involved in the exchanger. The direction of transport is determined by whether the energy provided by the entry of three sodium is greater than or less than the energy required to extrude one calcium. Page 3-10 (5)The energy released by sodium ions moving into the cell depends on the driving force on the sodium ions: sodium equilibrium potential - membrane potential (ENa -Vm ) The energy needed to move calcium ions out of the cell depends on the driving force on the calcium ion: calcium equilibrium potential - membrane potential (ECa - Vm ) If 3 (ENa -Vm ) > 2(ECa - Vm ), sodium ions move into the cell, while calcium ions move out of the cells. If 3 (ENa -Vm ) < 2(ECa - Vm ), sodium ions move out of the cell, while calcium ions move into the cells. (6) At some membrane potential, the energy for moving three sodium ions and the energy for moving one calcium ion will be equal. If we call this the reversal potential (Vr), then 3(ENa -Vr ) = 2(ECa - Vr ) or Vr = 3ENa -2 ECa At membrane potentials more negative than the reversal potential, sodium moves in and calcium moves out. At membrane potentials more positive, calcium moves in and sodium moves out. Chloride exchangers Intracellular chloride levels are kept low by two different chloride exchanger molecules. (1) The chloride bicarbonate exchanger moves chloride out of the cell and bicarbonate into the cell. This exchanger is driven by the movements of a proton out of the cell and a sodium ion into the cell down their electrochemical gradients. The chloride bicarbonate exchanger thus helps to regulate pH as well as chloride concentration in the cell. (2) The potassium chloride co-transporter moves chloride out of the cell by using the energy of potassium leaving the cell down its electrochemical gradient. ...
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