Presentation on theme: "Introduction Hodgkin and Huxley (1952):"— Presentation transcript:
1 IntroductionHodgkin 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 Isolation of Na+ and K+ Currents -9 mVMembrane Voltage-65 mV1Total CurrentMembrane Current-1Time after start of test pulse (msec)12345
3 Isolation of Na+ and K+ Currents -9 mVMembrane Voltage-65 mV+TTX1+KCurrentTotal CurrentMembrane Current-1Time after start of test pulse (msec)12345
4 Isolation of Na+ and K+ Currents -9 mVMembrane Voltage-65 mV+TTX1+KCurrentTotal CurrentMembrane Current+TEA+NaCurrent-1Time after start of test pulse (msec)12345
5 Voltage gates a channel Voltage sensors gate a channelVoltage gates a channelHille, B. Ionic Channels of Excitable Membranes 1984,1992,2001. (Sinauer Associates)
6 The region of the pore was rapidly localized by mutagenesis
7 A potassium channel subunit poreEXTRACELLULARS1S2S3S4S5S6INTRACELLULARTHE STRATEGYOut of the 8 positions we have looked at so far, 2 of the cysteine mutants, at positions 461, and 464 show rapid inhibition by intracellular Cd2+.NH2COO-
8 All voltage-gated channels possess conserved positively-charged “S4” residues
9 Four subunits or “repeats” assemble to form a complete channel EXTRACELLULARporeINTRACELLULARTHE STRATEGYOut of the 8 positions we have looked at so far, 2 of the cysteine mutants, at positions 461, and 464 show rapid inhibition by intracellular Cd2+.
10 The super-family of K+ channels is very diverse
11 The subunits assemble into tetramers, often with auxiliary b-subunits
12 The b-subunits often strongly change the properties of the channels
13 The superfamily of voltage-gated channels have similar four-fold symmetrical structures, with unique auxiliary subunits.
14 What are the conformational changes involved in gating and where (on the channel) do they occur? 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 Cd2+ (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.The next slide shows this approach graphically
15 S1 S2 S3 S4 S5 S6 NH2 COO- EXTRACELLULAR INTRACELLULAR THE STRATEGY Out of the 8 positions we have looked at so far, 2 of the cysteine mutants, at positions 461, and 464 show rapid inhibition by intracellular Cd2+.NH2COO-
16 An experiment showing “gated access”: Cysteine modifier,5 sec,“closed channels”Test pulses:+50 mVCysteine modifier,100 msec,“open channels”10.80.6Normalized current0.4RESULTSCd was applied to the patch for 25 sec while the patch was held at +10 mV, where the channels are mostly closed. This resulted in a 10% inhibition of current.In contrast, when Cd applied for 2 sec during a pulse to –110 mV, where the channels are maximally opened, 95% of the current was inhibited. The current recovered very little, even over the course of several minutes and with several long pulses to –110 mV.These results suggest that Cd gains access to a high affinity binding site ONLY when 464C channels are in the open state. To further explore this possibility, we looked at the apparent blocking rate at different voltages…0.212345Time (min)
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.InterpretationThe 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 The basic structure of a K+ channel pore is conserved across billions of years! 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 ).
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 b-subunit.Long et al., Science. 309:
20 Voltage-gated ion channels are often heavily modified in accord with their physiological functions Figure 6.3 Voltage-gated Ca2+ and K+ channels, key members of the voltage-gated ion channel family. (A) As with many other channels, CaV 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 α1 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) BKCa is an example of a K+ channel that has an additional domain sensitive to Ca2+. (A) from Arrikath and Campbell (Curr Op Neurobio 2003, pp ); (B) and (C) from Torres et al. (JBC 2007, pp ).
21 Crystal structure of a Bacterial K+ Channel KcsA 2xCrystal structure of a Bacterial K+ ChannelOUTCellmem-braneINNK+ ionsin poreC(Doyle, ..., MacKinnon, 1998)
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+ ionCavity ion surrounded by water? Stabilized by dipoles?
23 3.0 Ǻ resolution2.0 Ǻ resolutionCavity ion, surrounded by a cage of water
24 The mouth of the KcsA K channel KcsA MouthThe mouth of the KcsA K channelTurretP55OutervestibuleG56I60Q58A57Y82I60V84L81T85G79Y78G77V76Pore helixT75OuterhelixT75T74InnerhelixCentral cavity(Doyle, ..., MacKinnon, 1998)
25 The selectivity filter is intrinsically unstable,…providing for another possible “gate” High [K+]Low [K+]Zhou et al., Nature. 414:43-48.
27 Two completely opposite types of models to explain gating compete!! Jiang et al., Nature. 423:33-41.
28 A voltage-gated bacterial channel (KvAP) crystallized with an antibody fragment suggests the voltage sensor has a “paddle” motif!Fab fragmentsJiang et al., Nature. 423:33-41.
29 The “paddle motif” structure of KvAP Jiang et al., Nature. 423:33-41.
30 How might the voltage-sensor “paddle” close the pore??? Long et al., Nature. 450:
31 But proton currents (the “w-current”) suggest a “focused” electric field and that the “paddle” model is wrong!Starace and Bezanilla, Nature. 427:548-53
32 The “w-current” mechanism Tombola et al., Neuron. 45:379-88
33 FRET experiments also suggest the “paddle mode” must be wrong!! Chanda et al., Nature. 436,
34 Some K+ channels are turned on by neurotransmitters linked to G proteins 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.
35 The activity of some K+ channels is linked to the metabolism of the cell 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.
36 Voltage-gated calcium channels generate electrical signals Generate action potentialsStuart et al, 1997Back propagation of APsFatt and Ginsborg 1958Underlie oscillation of firingLlinas and Sugimori 1980Regulate firing patternLong and Connors (personal communication)In 1950s, Fatt and Katz demonstrated that voltage gated calcium channels generated sodium independent electrical spikes in crustecan muscle.
37 a1 b Ca2+ Voltage-gated calcium ion channels Excitation-secretion Excitation-contractionGene expressionNeurite outgrowthNeuronal excitabilityPacemakingCa2+
38 Voltage-gated calcium channels regulate various cellular functions Neurotransmitter releaseSecretionMuscle contractionActivity-dependent gene expressionCalcium levels inside cells are tightly controlledIntracellular levels are buffered at nMExtracellular calcium ~2 mM20,000-fold concentration gradientLargest driving force for any ionThis causes strong rectification of the current-voltage relationshipSecond messenger
39 The mechanism of selectivity for divalent Ca2+ channels has important differences from that of monovalent Na+ or K+ channelsFigure 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 Ca2 selectivity and ion permeation in L-type Ca2 channels [see comments]. Nature366, (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 ).