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Chapter 06FIG 1 Introduction Hodgkin and Huxley (1952): - Voltage-clamped squid giant axon - Found independent, selective Na +, K + conductances (ionic.

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Presentation on theme: "Chapter 06FIG 1 Introduction Hodgkin and Huxley (1952): - Voltage-clamped squid giant axon - Found independent, selective Na +, K + conductances (ionic."— Presentation transcript:

1 Chapter 06FIG 1 Introduction Hodgkin and Huxley (1952): - Voltage-clamped squid giant axon - Found independent, selective Na +, K + conductances (ionic basis for the action potential) - Conductances were “gated” by voltage: Na + and K + conductances increased at more positive voltages, turned off at negative voltages - Na + and K + conductances activated with different time courses - Na + current activation is rapid, and decreases rapidly during membrane depolarization (“inactivation”) - K + current activation is “delayed”, does not decrease - Suggested the presence of “gating particles”, charged elements that respond to voltage, linked to conductance increase; at least 3 for Na + conductance, 4 for K + conductance

2 Chapter 06FIG 2 Isolation of Na + and K + Currents -9 mV -65 mV Time after start of test pulse (msec) 012345 1 0 Membrane Voltage Membrane Current Total Current

3 Chapter 06FIG 3 Isolation of Na + and K + Currents -9 mV -65 mV Time after start of test pulse (msec) 012345 1 0 +TTX K + Current Membrane Voltage Membrane Current Total Current

4 Chapter 06FIG 4 Isolation of Na + and K + Currents -9 mV -65 mV Time after start of test pulse (msec) 012345 1 0 +TTX K + Current Membrane Voltage Membrane Current +TEA Na + Current Total Current

5 Chapter 06FIG 5 Voltage gates a channel Voltage sensors gate a channel Hille, B. Ionic Channels of Excitable Membranes 1984,1992,2001. (Sinauer Associates)

6 Chapter 06FIG 6 The region of the pore was rapidly localized by mutagenesis

7 Chapter 06FIG 7 EXTRACELLULAR INTRACELLULAR NH 2 COO- S1S2S3S5S6S4 A potassium channel subunit pore

8 Chapter 06FIG 8 All voltage-gated channels possess conserved positively-charged “S4” residues

9 Chapter 06FIG 9 EXTRACELLULAR INTRACELLULAR Four subunits or “repeats” assemble to form a complete channel pore

10 Chapter 06FIG 10 The super-family of K + channels is very diverse

11 Chapter 06FIG 11 The subunits assemble into tetramers, often with auxiliary  -subunits

12 Chapter 06FIG 12 The  -subunits often strongly change the properties of the channels

13 Chapter 06FIG 13 The superfamily of voltage-gated channels have similar four-fold symmetrical structures, with unique auxiliary subunits.

14 Chapter 06FIG 14 Strategy: Focusing on the S6 transmembrane region, each residue was substituted (one at a time) with cysteine; The cysteine mutants were then probed with cysteine-modifying (MTS) reagents, or Cd 2+ (which binds to cysteines), to test their reactivity when the channels are held open or closed. The reactivity was measured functionally as a change in the size of the current through the channels. What are the conformational changes involved in gating and where (on the channel) do they occur? The next slide shows this approach graphically


16 Chapter 06FIG 16 0 0.2 0.4 0.6 0.8 1 012345 Time (min) Normalized current Cysteine modifier, 5 sec, “closed channels” Test pulses: +50 mV Cysteine modifier, 100 msec, “open channels” An experiment showing “gated access”:

17 Chapter 06FIG 17 Major findings:  Residues toward the extracellular end of S6 could be modified only when the channels were held open, at depolarized voltages.  Residues at the intracellular end of S6 could be modified regardless of whether the channels were open or closed. Interpretation  The S6 region probably lines the pore, and access to this region is controlled by a gate.  Confirmed and expanded on the hypothesis of Armstrong: localized the residues that line the cavity.  (Other experiments and results in the paper hinted that part of the S6 may itself form the gate)

18 Chapter 06FIG 18 Figure 6.1 Examples of ion channel pores from various potassium channels. (A) A water-filled cavity is formed from four protein subunits, two of which are shown in the bacterial potassium channel, KcsA. The cavity creates a passageway through which ions can flow across the membrane, into or out of the cell. (B) The unique amino acid sequence of each family of channels allows it to selectively filter out particular ions. In the case of KcsA, K + but not Na + ions are allowed to pass through the selectivity filter, even though K + ions are bigger than Na + ions. S1-4 refers to the four K + ion-binding sites in the selectivity filter, each composed of eight oxygen atoms from the TVGYG signature motif. (C) Pore-region sequence alignments of five structurally known potassium channels are shown with the GYG signature motif boxed in magenta and other highly conserved regions labeled in black. (D) Structural comparison of the pore regions from the same five potassium channels. (A) and (B) adapted from Lockless et al. (PloS 2007, p. e121); (C) and (D) from Shrivastava and Bahar (Biophys J 2006, pp. 3929-3940). The basic structure of a K + channel pore is conserved across billions of years!

19 Chapter 06FIG 19 The crystal structure of a mammalian voltage-gated K + channel reveals a “modular” assembly of a voltage-sensor domain, a pore domain, a tetramerization domain, and a  -subunit. Long et al., Science. 309:897-903

20 Chapter 06FIG 20 Figure 6.3 Voltage-gated Ca 2+ and K + channels, key members of the voltage-gated ion channel family. (A) As with many other channels, Ca V channels consist of many protein domains that allow the channel to be regulated by a variety of extra- and intracellular signals, in addition to voltage sensitivity through the α 1 subunit. (B) Voltage-gated K + channels consist of four α subunits that together form a pore for the passage of ions, as well as a cytoplasmic β subunit. (C) BK Ca is an example of a K + channel that has an additional domain sensitive to Ca 2+. (A) from Arrikath and Campbell (Curr Op Neurobio 2003, pp. 298-307); (B) and (C) from Torres et al. (JBC 2007, pp. 24485-24489). Voltage-gated ion channels are often heavily modified in accord with their physiological functions

21 Chapter 06FIG 21 Crystal structure of a Bacterial K + Channel (Doyle,..., MacKinnon, 1998) KcsA 2x C N OUT IN Cell mem- brane K + ions in pore

22 Chapter 06FIG 22 The KcsA potassium channel (front and back subunits removed for clarity) K+ ions, coordinated by carbonyl oxygens in the “selectivity filter” Large cavity, contains a K + ion Cavity ion surrounded by water? Stabilized by dipoles?

23 Chapter 06FIG 23 3.0 Ǻ resolution 2.0 Ǻ resolution Cavity ion, surrounded by a cage of water

24 Chapter 06FIG 24 KcsA Mouth Outer vestibule P55 A57 V84 G79 Y78 G77 V76 T75 T74 I60 T85 Q58 G56 L81 Y82 Turret Central cavity Inner helix Pore helix Outer helix (Doyle,..., MacKinnon, 1998) The mouth of the KcsA K channel

25 Chapter 06FIG 25 Low [K+] High [K+] The selectivity filter is intrinsically unstable,…providing for another possible “gate” Zhou et al., Nature. 414:43-48.

26 Chapter 06FIG 26 Early depictions of possible gating mechanisms

27 Chapter 06FIG 27 Two completely opposite types of models to explain gating compete!! Jiang et al., Nature. 423:33-41.

28 Chapter 06FIG 28 A voltage-gated bacterial channel (KvAP) crystallized with an antibody fragment suggests the voltage sensor has a “paddle” motif! Fab fragments Jiang et al., Nature. 423:33-41.

29 Chapter 06FIG 29 The “paddle motif” structure of KvAP Jiang et al., Nature. 423:33-41.

30 Chapter 06FIG 30 How might the voltage-sensor “paddle” close the pore??? Long et al., Nature. 450:376-382.

31 Chapter 06FIG 31 But proton currents (the “  -current”) suggest a “focused” electric field and that the “paddle” model is wrong! Starace and Bezanilla, Nature. 427:548-53

32 Chapter 06FIG 32 The “  -current” mechanism Tombola et al., Neuron. 45:379-88

33 Chapter 06FIG 33 FRET experiments also suggest the “paddle mode” must be wrong!! Chanda et al., Nature. 436, 852-856.

34 Chapter 06FIG 34 Figure 6.5 G-protein-activated inwardly rectifying potassium channels (Kir3) are activated by direct interaction with the βγ subunits of G protein. L represents the ligand for the G-protein-coupled receptor with seven transmembrane segments, e.g., the parasympathetic transmitter acetylcholine for slowing the heart rate or the inhibitory transmitter GABA for generating the slow inhibitory postsynaptic potential in the central nervous system. Some K + channels are turned on by neurotransmitters linked to G proteins

35 Chapter 06FIG 35 Figure 6.6 The ATP-sensitive potassium channels contain four pore-lining α subunits (Kir6) and four regulatory β subunits (SUR). SUR is a member of the ATP-binding cassette (ABC) family and contains two nucleotide-binding (NB) domains. ATP acts on Kir6 to inhibit the channel whereas Mg-ADP acts on SUR to activate the channel. Sulfonylurea (SU) drugs that inhibit the channel and KCO compounds that activate the channel also act on SUR. The activity of some K + channels is linked to the metabolism of the cell

36 Chapter 06FIG 36 Voltage-gated calcium channels generate electrical signals Fatt and Ginsborg 1958 Generate action potentials Underlie oscillation of firing Llinas and Sugimori 1980 Regulate firing pattern Long and Connors (personal communication) Stuart et al, 1997 Back propagation of APs

37 Chapter 06FIG 37   Voltage-gated calcium ion channels Excitation-secretion Excitation-contraction Gene expression Neurite outgrowth Neuronal excitability Pacemaking Ca 2+

38 Chapter 06FIG 38 Neurotransmitter release Secretion Muscle contraction Activity-dependent gene expression Voltage-gated calcium channels regulate various cellular functions Calcium levels inside cells are tightly controlled Intracellular levels are buffered at 100-200 nM Extracellular calcium ~2 mM 20,000-fold concentration gradient Largest driving force for any ion This causes strong rectification of the current-voltage relationship

39 Chapter 06FIG 39 Figure 6.12 Dependence of voltage-gated calcium channel ion selectivity on calcium concentration. (A) In the presence of sodium ions and varying concentration of calcium ions, calcium channels are permeable to sodium ions at submicromolar calcium concentration. At submillimolar calcium concentration, a calcium ion occupies one binding site in the channel and blocks sodium permeation. At still higher calcium concentration, calcium may occupy multiple binding sites; the presence of multiple calcium ions in the same channel pore allows them to dissociate from the binding site more readily and pass through the channel. Adapted from Almers and McCleskey (1984). (B) The affinity of the calcium-binding site as indicated by the blocking action of calcium on lithium permeation is reduced by substituting a glutamate in the P loop with glutamine. WT, wild-type calcium channel. I, II, III, and IV indicate glutamine substitution in the first, second, third, and fourth repeats of the channel. I + IV indicates double mutations in the first and fourth repeats. (C) How the ring of four glutamate residues in the calcium channel pore might bind one or two calcium ions. (B) and (C) are adapted with permission from Macmillan Publishers Ltd. Yang, J., Ellinor, P.T., Sather, W.A., Zhang, J.F., and Tsien, R.W. (1993). Molecular determinants of Ca 2 selectivity and ion permeation in L-type Ca 2 channels [see comments]. Nature366, 158-161. (D) Glutamate substitution of lysine 1422 of the P loop in the third repeat of voltage-gated sodium channels causes the mutant channel to behave like a calcium channel. (E) Alignment of the P loop sequences for each of the four repeats of the voltage-gated sodium channels and calcium channels. (D) and (E) are adapted with permission from Macmillan Publishers Ltd. Heinemann et al. (Nature 1992, pp. 441-443). The mechanism of selectivity for divalent Ca 2+ channels has important differences from that of monovalent Na + or K + channels

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