Membrane Channels The Cell Membrane is Selective • Criteria for passage through the phospholipid bilayer: 1. Hydrophobic 2. Net zero charge 3. Nonpolar 4. Size is also a consideration • Chemicals that will NOT pass through the phospholipid bilayer: 1. Hydrophillic 2. Charged, ionic 3. Polar 4. Size is also a consideration • But, using the preceding criteria, many substances vital to cellular function (e.g., ions) and survival (e.g., glucose) will not gain entry into the cell! • So, nature has come up with channels, which selectively allow certain substances to gain entry into the cell, even though they do not meet the criteria on the preceding slide. The Many Functions of Membrane Proteins Outside Plasma Membrane Inside Transported Cell Surface Identity Marker Enzyme Activity Cell Adhesion Cell Surface Receptor Attachment to the Cytoskleton Channels are Vital Without channels it is energetically unfavorable to move ions across a membrane – 1. the phospholipid bilayer is ~6-8 nm thick. 2. the hydrophilic head of the phospholipid molecule projects toward the cytoplasm or the extracellular fluid. 3. the hydrophobic tails of the phospholipid molecules project toward each other. Transmembrane Transport • • • a) communication among neurons neural systems: * action potential * synaptic signaling b) receptor – brain communication heart muscle signaling and regulatory processes Channel malfunction Cystic fibrosis Epilepsy Diabetes Migraines Neurotoxins For the Cation to Move Through the Phospholipid Bilayer…, 1) It must lose its waters of hydration so that it is not so huge and charged; requires energy to break attractive forces between the ion and the waters. 2) Energy is also required to move a charged highly hydrophilic particle into the highly hydrophobic area of the lipid bilayer that contains the “tails” of the phospholipid molecules. 3) Based on thermodynamic calculations, so much energy would be required for this process that it would never occur. Channels are thermodynamically similar to Enzymes in that the former lower the Activation Energy required to move ions across the Membrane Transition State The rate of the reaction is determined by the energy of activation, the energy input required to produce the transition state. The uncatalyzed reaction requires a higher activation energy than the catalyzed one does. So, the latter runs more quickly. A+B Reactants (substrates) AB transition state C+D products There is no difference in free energy (ΔG) between uncatalyzed and catalyzed reactions. The ΔG is the thermodynamic driving force for the reaction and determines the direction of the reaction. Ion Flow Across the Membrane • A Chemical can move across the membrane through one of two ways: 1. Movement through the phospholipid bilayer. 2. Movement through a H2O-filled protein channel. Channels are thermodynamically similar to Enzymes in that the former lower the Activation Energy required to move ions across the Membrane The rate of diffusion is determined by the “energy of activation”, the energy input required to produce the “transition state”. (Remove H2Os of hydration and/or move into hydrophobic environment.) Movement through a channel only requires shedding of waters of hydration (energy input would be infinite to move through the bilayer). So. Diffusion occurs more quickly. ΔG determines this!! Transition State Change in free energy is the thermodynamic driving force for diffusion and determines the direction of ion movement A+B Reactants (substrates) AB transition state C+D products What do we know about the structure of gated ion channels? A. Biochemical Information – 1. 2. 3. 4. MWs range from 25-250 kDal. They are integral membrane glycoproteins. They usually consist of 2 or more subunits. The genes that code for the proteins have been isolated, cloned and sequenced. These sequences have been grouped into 6-7 protein families. 5. The primary (amino acid) sequences of these channels is known. Use of the Hydrophobicity Plots 1) Propose 3-D structures of the channels 2) Propose functions for specific regions of the channel proteins Amino Acid Sequence Enables Ion Channel Structure Determination Many Techniques Can be Used to Test the Proposed Functions of Portions of the Channel Proteins 1) Sequence Homologies – used to determine which portions of primary sequences of ion channels are the same/very similar a) Same channel from several different species – what is conserved must be critical to channel function e.g., Ach-gated channel – Ach receptor portion of the channel is highly conserved Many Techniques Can be Used to Test the Proposed Functions of Portions of the Channel Proteins 1) Sequence Homologies – used to determine which portions of primary sequences of ion channels are the same/very similar (continued) b) Different channels with the same basic function from many tissues in one species – what is conserved must be critical to channel function e.g., Voltage gated channels (K+, Na+, Ca2+) – all have a presumed membrane-spanning region with charged AAs at each third position (voltage sensor?), while ligand-gated channels lack this structure Many Techniques Can be Used to Test the Proposed Functions of Portions of the Channel Proteins 2) Immunocytochemistry – a) raise antibodies to a portion of the molecule thought to be on the intracellular or extracellular surface of the membrane b) Incubate neurons with the labeled antibodies c) Do antibodies bind to intact neurons? (Note: antibodies are too large to fit into a channel) Yes – sequence is on extracellular surface No – sequence may be in pore or on intracellular surface d) Next step: lyse cells and repeat exp. to see if there is binding, i.e. is it an intracellular sequence? Many Techniques Can be Used to Test the Proposed Functions of Portions of the Channel Proteins 3) Site-directed mutagenesis – use molecular biology techniques to modify specific regions of a channel with a predicted function – does modification alter channel function in a predicted fashion? 4) Chimaeric Channel Construction – construct a mutant channel from sequences from 2 or more channel genes – which “parent” channel does the mutant resemble? •Most channels have this basic structure: multimeric (quarternary structure), membranespanning, and, by definition, have a pore running longitudinally through the structure. •Vary in the number of subunits and complexity. Remember your amino acids? • Primary, secondary, and tertiary structures of proteins. • In addition, recall that multimeric proteins are formed from the attraction of individual subunits, forming the quarternary structure. • Recall the structure and ionization of the each of the amino acid side-chains (R). -It wouldn’t hurt if you reviewed what a pI is. The amino acid side –chains (R) •The primary amino acid sequence and higher –order structures determine the channel topology. Interior of the channel will be lined with hydrophilic amino acids. Exterior of the channel will be lined with hydrophobic amino acids. Selectivity Filter • Many channels are selective for only 1 or 2 different chemicals (ions, sugars, etc.). • The K+ channel has such a filter, which is a narrow region towards the extracellular surface of the membrane. • Two K+ ions can occupy the selectivity filter simultaneously, with a third in a H2O-filled cavity deeper in the pore. Proposed Mechanisms for Channel Ion Selectivity Non-specific cation channel, i.e. little selectivity other than for cations Ach receptor channel - 6.5 A in diameter 10-20 X more Na+ than K+ 100 X more K+ than Na+ Voltagegated Na+ channel 4 A in diameter Voltagegated K+ channel – 3.3 A in diameter Proposed Mechanisms for Channel Ion Selectivity by Channels: Ionic size Non-specific cation channel, i.e. little selectivity other than for cations Ach receptor channel - 6.5 A in diameter If ionic size explains channel selectivity, why is the K+ channel so selective for K+ since Na+ is smaller? 10-20 X more Na+ than K+ Voltagegated Na+ channel - 4 A in diameter Nonhydrated K+ ion = 2.7 A in diameter 100 X more K+ than Na+ Voltage-gated K+ channel – 3.3 A in diameter Nonhydrated Na+ ion = 1.9 A in diameter Proposed Mechanisms for Ion Selectivity by Channels: Ionic size Non-specific cation channel, i.e. little selectivity other than for cations Ach receptor channel - 6.5 A in diameter Modified Model = perhaps channels select based on hydrated ionic radius? 10-20 X more Na+ than K+ Voltagegated Na+ channel - 4 A in diameter 100 X more K+ than Na+ Voltage-gated K+ channel – 3.3 A in diameter Hydrated Na+ ion = 3.3-4 A in diameter Hydrated K+ ion = 3.3 A in diameter (K+ is larger, has a lower charge density and so attracts fewer waters of hydration.) Proposed Mechanisms for Ion Selectivity by Channels: Ionic size The modified model explains K+ channel selectivity, i.e. the hydrated K+ just fits into the channel and the hydrated Na+ is too big to fit. However, how do we explain the +/- sodium channel selectivity? A selectivity filter exists inside the channel Proposed Mechanisms for Ion Selectivity by Channels: Ionic size Sodium recognition site = selectivity filter Na+ Na+ How might it work? Similar to enzymes, but much faster? Evidence for a Selectivity Filter If channels are simple resistors, than movement through an open channel should be a function of the concentration gradient for the ion across the membrane Rate of ion movement = a x [ion]I/[ion]o (current flow = diffusion) Linear relationship with slope = a Evidence for a Selectivity Filter Observed data for Na+ channel Unitary current (pa) = recordings from single channels Expected data External [Na+] mM Evidence for a Selectivity Filter Data for voltage-gated Na+ channel do not fit the model of a channel as a simple resistor in the membrane. Instead, the current flow through the Na+ channel plateaus or “saturates” at high [Na+]. This relationship looks like what happens to an enzyme at high [substrate]. Perhaps some channels select ions based on the same biochemical mechanisms used by enzymes to select their substrates? In the end, the final determinations of channel gating mechanisms and ion selectivities will come from Xray crystallography of the purified channels. Crystal Structure of the K+ Channel from above and from the side The K+ channel is structure such that a very narrow tube through the inverted cone shape allows for only 50 H2O molecules and only 2 K+ in succession. Because they strongly repel each other, when one enters, one will be forced out. Methods for Studying Ion Channels - 1 Biochemistry – agonist, antagonist or drug binding – isolation and purification – reconstitution Molecular biology – sequencing, cloning, mutagenesis Structural biology – microscopy, crystallography, NMR, ... Methods for Studying Ion Channels - 2 Electrophysiology – tissue slice – extracellular recording – intracellular recording – whole-cell recording – single channel recording Biochemistry – radioactive ion flux Voltage clamp Current clamp Concentration jump Cloning via Protein Purification Electric eel Pure Na channel proteins Protein Purification Na channel proteins Microsequence Oligonucleotide probe with sequence corresponding to aa sequence Design probe Hybridize to cDNA library containing Na channel cDNA sss sss sss Synthesize cDNA library containing Na channel cDNA (s) sssss sssss ssssss Amino acid sequence of small region of Na channel protein Isolate and sequence Na channel cDNA Deduce protein sequence Isolate mRNAs including one encoding Na channel AA sequence of entire Na channel protein • Positional cloning – Shaker flies Normal Shaker • Cloning by sequence homology - Use the same strategy as that used in preceding slide, except that the sequence on which the cloning is based comes not from the purified protein, but rather from the alreadyisolated cDNA. Positional Cloning Enabled Determination of the Shaker K Channel Heterologous Expression Systems Typical Ion Channels with Known Structure: K+ channel (KCSA) Acetylcholine receptor M2 transmembrane segment Types of ion channels: Simple pores (GA, GAP junctions) Substrate gated channels (Nicotinic receptor) Voltage-gated channels (K-channels) Pumps (ATP-synthase, K+,Na+-ATPase) • Ion Channels • • • ion channels in the PM of neurons and muscles contributes to their excitability when open - ions move down their concentration gradients channels possess gates to open and close them two types: gated and non-gated 1. Leakage (non-gated) or Resting channels: are always open, contribute to the resting potential -nerve cells have more K+ than Na+ leakage channels -as a result, membrane permeability to K+ is higher -K+ leaks out of cell - inside becomes more negative -K+ is then pumped back in 2. Gated channels: open and close in response to a stimulus A. voltage-gated: open in response to change in voltage - participate in the AP B. ligand-gated: open & close in response to particular chemical stimuli (hormone, neurotransmitter, ion) C. mechanically-gated: open with mechanical stimulation Types of Biochemical Mechanisms that Open and Close Channels (Cont’d) • Nt or hormone binding to receptor causes a 2nd messenger to activate a protein kinase that phosphorylates a channel and thus opens it. • Changes in membrane potential. • Membrane deformation (e.g., mechanical pressure). • Selectivity by charge (i.e., positively lined pore allows anions through; negatively lined pore allows cations through). Na+ Channels have Gates At rest, one is closed (the activation gate) and the other is open (the inactivation gate). Suprathreshold depolarization affects both of them. The resting potential, recall, is generated mainly by open “resting”, non-gated K+ channels -the number of K+ channels dramatically outnumbers that of Na+ -however, there are a few Na leak channels along the axonal membrane AXON ECF Channel Gating Mechanisms AChR: Proposed gating mechanism (Unwin, 1995) Closed Open Channel Families • • • • • • Voltage-gated Extracellular ligand-gated Intracellular ligand-gated Inward rectifier Intercellular Other Voltage-gated • sodium: I, II, III, µ1, H1, PN3 • potassium: KA, Kv (1-5), Kv(r), Kv(s),KSR, BKCa, IKCa, SKCa, KM, KACh • calcium: L, N, P, Q, T • chloride: ClC-0 - ClC-8 Extracellular ligand-gated • nicotinic ACh (muscle): 2 (embryonic), 2 (adult) • nicotinic ACh (neuronal): (2-10), (2-4) • glutamate: NMDA, kainate, AMPA • P2X (ATP) • 5-HT3 • GABAA: (1-6), (1-4), (1-4), , , (1-3) • Glycine Intracellular ligand-gated • leukotriene C4-gated Ca2+ • ryanodine receptor Ca2+ • IP3-gated Ca2+ • IP4-gated Ca2+ • Ca2+-gated K+ • Ca2+-gated nonselective cation Ca2+-gated Cl– cAMP cation cGMP cation cAMP chloride ATP Cl– volume-regulated Cl– arachidonic acidactivated K+ • Na+-gated K+ • • • • • • • Inward rectifier • Kir – – – – – – – 1.1-1.3 2.1-2.4 3.1-3.5 4.1-4.2 5.1 6.1-6.2 7.1 Intercellular • Gap junction channels – – – – – – – – Cx26 Cx32 Cx37 Cx40 Cx43 Cx45 Cx50 Cx56 Other • • • • • Mechanosensitive Mitochondrial Nuclear Aquaporins Vesicular (synaptophysin) G-protein linked receptors coupled to ion channels • • • • • • • • • • • • • • • Acetylcholine (muscarinic) Adenosine & adenine nucleotides Adrenaline & noradrenaline Angiotensin Bombesin Bradykinin Calcitonin Cannabinoid Chemokine Cholecystokinin & gastrin Dopamine Endothelin Galinin GABA (GABAB) Glutamate (quisqualate) • • • • • • • • • • • • • • • Histamine 5-Hydroxytryptamine (1,2) Leukotriene Melatonin Neuropeptide Y Neurotensin Odorant peptides Opioid peptides Platelet-activating factor Prostanoid Protease-activated Tachykinins Taste receptors VIP Vasopressin and oxytocin Structure of the AChR at 4.6Å Miyazawa et al, (1999) (left figure was modified) Outside Cell Na+ Ca2+ Cl- K+ Inside Cell Schematic model of AChR ligand binding site NH2 S -S- - Y W ACh or ant agonist C C based on Arias, 1997 NH2 Y W T D Y E . -S-S - empty site in ACh binding protein Brejc, 2001 filled site in ACh binding protein Brejc, 2001 K Channel Structure at 3Å Resolution Doyle et al, 1998 Patch Clamp Recording Technique A B C D E el ect r ode channel cel l cel l- at t ached pat ch whol e- cel l out si de- out pat ch Sizing Up Ion Channels Gating and Permeation What are the Biochemical Changes that Lead to Channel Gating (Opening or Closing)? Gating involves some type of conformational change in the protein, but other than that there are few definitive answers to the question. However, there are several general proposed models for gating. Types of Biochemical Mechanisms that Open and Close Channels • Conformational change occurs in a discrete area of the channel, leading to it opening. • The entire channel changes conformation (e.g., electrical synapses). • Ball-and-chain – type mechanism. • Nt or hormone binding causes the channel to open. What are the Biochemical Changes that Lead to Channel Gating (Opening or Closing)? A. A conformation change occurs in a discrete portion of the channel Most popular model, but currently no evidence for it! closed opened What are the Biochemical Changes that Lead to Channel Gating (Opening or Closing)? B. Global change occurs in the channel Evidence that electrical synapses = gap junctions function in this manner closed opened What are the Biochemical Changes that Lead to Channel Gating (Opening or Closing)? C. A blocking portion of the channel changes position Evidence that voltagegated channels function in this manner closed opened What Types of Stimuli Can Lead to Gating? A. Binding of Chemicals = Ligand-gated or Chemically-gated Channels 1) Extracellular surface – neurotransmitter or hormone binding causes opening of an ion channel Neurotransmitter or hormone Binds to Opened channel receptor protein What Types of Stimuli Can Lead to Gating? A. Binding of Chemicals = Ligand-gated or Chemically-gated Channels (continued) 2) Intracellular surface – a second messenger is activated by neurotransmitter or hormone binding to a receptor Neurotransmitter or hormone Binds to 1) Binds directly to a channel and opens/closes it receptor protein 2nd messenger 2) Activates a protein kinase that phosphorylates the channel and opens/closes it What Types of Stimuli Can Lead to Gating? B. Change in Membrane Potential = Voltage-gated channels C. Membrane Deformation = Mechanically-gated channels (ex. Sensory system where membrane stretch or pressure on the membrane is transmitted to the channel via the cytoskeleton?) Pressure applied What are the Proposed Mechanisms for Ion Selectivity by Channels? A. Channels can select by charge ClAnion selective channel lined with positively charged AAs + + + + + + ClK+ Cation selective channel lined with negatively charged AAs - - - - - - K+ What are the Proposed Mechanisms for Ion Selectivity by Channels? B. Channels can select based on ionic size – Experimental approach = 1) sequence data were used to propose 3-D structures of the channels and to estimate the pore diameters 2) ionic selectivities were determined using electrophysiological techniques A Closer Look at Gating: Kinetics of Gating Channel Gating: Two State Model Closed k o k c dClosed k o Closedk c Open dt Open dOpen k o Closedk c Open dt Channel Gating: 2 State Simulations Slow kinetics: ko=0.5/ms kc=0.5/ms 0 1 2 Time (ms) Fast kinetics: ko=5/ms kc=5/ms 3 4 0 0 1 2 Time (ms) 1 2 Time (ms) Superposition Open Prob 1.0 0.8 0.6 0.4 0.2 0.0 3 4 3 4 Gating Exercise Closed average closed t ime 1 ko k o k c Open st eady st at e p average open t ime 1 kc open onset t ime k o ko kc 1 ko kc Predictions example 1 ko (1/ms) 0.5 kc (1/ms) 0.5 2 5.0 5.0 3 5.0 0.5 4 0.5 5.0 average closed time (ms) 2.0 average open time (ms) 2.0 steadystate p open 0.5 onset time (ms) 1.0 Channel Gating: More 2 State Simulations High popen: ko=5/ms kc=0.5/ms 0 1 2 Time (ms) 3 4 Low popen: ko=0.5/ms kc=5/ms 0 1 2 Time (ms) Superposition Open Prob 1.0 0.8 0.6 0.4 0.2 0.0 0 1 2 Time (ms) 3 4 3 4 Channel Gating: 3 State Model More realistic model V-gated Na channels open channel blockers Closed k o k c Open k +i k -i Inactive dClosed k o Closed k c Open dt dOpen k o Closed k c Open k Open k Inactive +i -i dt dInactive dt k +i Open k Inactive -i Channel Gating: 3 State Simulation k k Closed o k c Open +i ko = 0.1/ms kc =0.5/ms k+i = 1/ms k-i = 5/ms Inactive k -i Open Closed 0 10 20 30 time (ms) 40 50 Burst Current Open Inactive Closed "Supervision" 4 6 8 10 time (ms) 12 14 Channel Gating: 3 State Superposition 1.0 Popen Open Prob 0.8 0.6 0.4 0.2 0.0 0 10 20 30 time (ms) ko = 0.5/ms kc =0.005/ms k+i = 0.25/ms k-i = 0.025/ms k+i = 0/ms k-i = 0/ms 40 50 Channel Gating Mechanisms (1) The “Ball and Chain” model of channel inactivation gating At depolarized potentials, the ball is more stable at the blocking conformation. Channel Gating Mechanisms (2) AChR: Open (white) & Closed (blue) conformations (Unwin, 1995) Channel Gating Mechanisms (3) AChR: Proposed gating mechanism (Unwin, 1995) Channel Permeation - Nernst Consider a cell containing a high concentration of KCl External [KCl] is low Consider the cell membrane to be permeable to K+ only. K KK+++ K KK+++ ++ K K -- - Cl Cl Cl -- Cl Cl initial condition –– -- - Cl Cl Cl –– ++ K K –– -Cl –– Cl –– after some diffusion Vr is known as the: Nernst potential reversal potential zero current potential equilibrium potential –– –– –– ++ –– –– K –– –– K –– –– Cl Cl Cl Cl-- - –– Cl –– –– –– K KK+++ equilibrium c c Vr = RT ln co 58mV log co z zF i i Channel Permeation: Examples 58 mV co Vr = log c z i 58 mV 5 mM VK = log 86 mV 150 mM +1 [K]o =5 mM, [K]i=150 mM Ion z co (mM) ci (mM) Vr (mV) K +1 5 150 -86 Na 150 15 Cl 120 10 Ca 2 0.0001 Channel Permeation: GHK Goldman-Hodgkin-Katz Voltage Equation Cell membrane potential depends on ion gradients and ionic permeabilities (Pi) Vm = 58 mV log PK K o PNaNa o PClCli PK Ki PNaNa i PClClo 1. Neuron at rest (ci, co from previous slide) PK=100, PNa=5, PCl=10 Vm= 63 mV 2. Neuron during an action potential PK=100, PNa=500, PCl=10 Vm=___ Channel Permeation: Ohm's Law Current (flow) is equal to voltage (driving force) times conductance (1/resistance) I = (V-Vr) G Current (I) is measured in amps (pA, nA, µA) Voltage (V) is measured in volts (mV) Conductance (G) is measured in Siemens (pS, nS) Channel Permeation: Turnover How much is a picoamp of current? 1 ion 7 ions/s 1pA 10 12 A 10 12 Coul 10 s 1.6 10 19 Coul Ion channels are enzymes that catalyze the flow of ions across cell membranes. The catalytic rate is on the order of 107 per second. Channel Permeation: IV Curves a) V-independent conductance: I = (V-Vr) G, G = N popen 1000 1.0 500 Current (pA) Open Probability 0.8 0.6 0.4 0.2 0.0 -100 -50 0 50 100 0 -500 -1000 -100 -50 Voltage (mV) 0 Voltage (mV) N=200, = 50 pS, V =0 r 50 100 Channel Permeation: IV Curves b) V-dependent conductance I = (V-Vr) G(V) G = N popen(V) 1000 1.0 500 Current (pA) Open Probability 0.8 0.6 0.4 0.2 0.0 -100 -50 0 50 100 0 -500 -1000 -100 -50 Voltage (mV) 0 Voltage (mV) N=200, = 50 pS, V =+50 mV r 50 100