Post on 19-Jan-2016
Chapter 06 FIG 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
Chapter 06 FIG 2
Isolation of Na+ and K+ Currents
-9 mV
-65 mV
Time after start of test pulse (msec)
0 1 2 3 4 5
1
0
-1
Membrane Voltage
Membrane Current
Total Current
Chapter 06 FIG 3
Isolation of Na+ and K+ Currents
-9 mV
-65 mV
Time after start of test pulse (msec)
0 1 2 3 4 5
1
0
-1
+TTXK+ Current
Membrane Voltage
Membrane Current
Total Current
Chapter 06 FIG 4
Isolation of Na+ and K+ Currents
-9 mV
-65 mV
Time after start of test pulse (msec)
0 1 2 3 4 5
1
0
-1
+TTXK+ Current
Membrane Voltage
Membrane Current
+TEANa+ Current
Total Current
Chapter 06 FIG 5
Voltage gates a channel
Voltage sensors gate a channel
Hille, B. Ionic Channels of Excitable Membranes 1984,1992,2001. (Sinauer Associates)
Chapter 06 FIG 6
The region of the pore was rapidly localized by mutagenesis
Chapter 06 FIG 7
EXTRACELLULAR
INTRACELLULAR
NH2
COO-
S1 S2 S3 S5 S6S4
A potassium channel subunit
pore
Chapter 06 FIG 8
All voltage-gated channels possess conserved positively-charged “S4” residues
Chapter 06 FIG 9
EXTRACELLULAR
INTRACELLULAR
Four subunits or “repeats” assemble to form a complete channel
pore
Chapter 06 FIG 10
The super-family of K+ channels is very diverse
Chapter 06 FIG 11
The subunits assemble into tetramers, often with auxiliary -subunits
Chapter 06 FIG 12
The -subunits often strongly change the properties of the channels
Chapter 06 FIG 13
The superfamily of voltage-gated channels have similar four-fold symmetrical structures, with unique auxiliary subunits.
Chapter 06 FIG 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 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.
What are the conformational changes involved in gating and where (on the channel) do they occur?
The next slide shows this approach graphically
Chapter 06 FIG 15
EXTRACELLULAR
INTRACELLULAR
NH2
COO-
S1 S2 S3 S5 S6S4
Chapter 06 FIG 16
0
0.2
0.4
0.6
0.8
1
0 1 2 3 4 5Time (min)
Norm
aliz
ed c
urr
ent
Cysteine modifier,
5 sec,“closed
channels”
Test pulses:+50 mV Cysteine modifier,
100 msec,“open channels”
An experiment showing “gated access”:
Chapter 06 FIG 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)
Chapter 06 FIG 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!
Chapter 06 FIG 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
Chapter 06 FIG 20
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. 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
Chapter 06 FIG 21
Crystal structure of a Bacterial K+ Channel
(Doyle, ..., MacKinnon, 1998)
KcsA 2x
C
N
OUT
IN
Cellmem-brane
K+ ionsin pore
Chapter 06 FIG 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?
Chapter 06 FIG 23
3.0 Ǻ resolution 2.0 Ǻ resolution
Cavity ion, surrounded by a cage of water
Chapter 06 FIG 24
KcsA Mouth
Outer vestibule
P55
A57 V84
G79
Y78
G77
V76
T75
T75T74
I60I60
T85
Q58
G56
L81
Y82
Turret
Central cavityInnerhelix
Pore
hel
ix
Outerhelix
(Doyle, ..., MacKinnon, 1998)
The mouth of the KcsA K channel
Chapter 06 FIG 25
Low [K+] High [K+]
The selectivity filter is intrinsically unstable,…providing for another possible “gate”
Zhou et al., Nature. 414:43-48.
Chapter 06 FIG 26
Early depictions of possible gating mechanisms
Chapter 06 FIG 27
Two completely opposite types of models to explain gating compete!!
Jiang et al., Nature. 423:33-41.
Chapter 06 FIG 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.
Chapter 06 FIG 29
The “paddle motif” structure of KvAP
Jiang et al., Nature. 423:33-41.
Chapter 06 FIG 30
How might the voltage-sensor “paddle” close the pore???
Long et al., Nature. 450:376-382.
Chapter 06 FIG 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
Chapter 06 FIG 32
The “-current” mechanism
Tombola et al., Neuron. 45:379-88
Chapter 06 FIG 33
FRET experiments also suggest the “paddle mode” must be wrong!!
Chanda et al., Nature. 436, 852-856.
Chapter 06 FIG 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
Chapter 06 FIG 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
Chapter 06 FIG 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
Chapter 06 FIG 37
Voltage-gated calcium ion channels
Excitation-secretionExcitation-contractionGene expressionNeurite outgrowthNeuronal excitabilityPacemaking
Ca2+
Chapter 06 FIG 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
Chapter 06 FIG 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 Ca2 selectivity and ion permeation in L-type Ca2 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 Ca2+ channels has important differences from that of monovalent Na+ or K+ channels