Ion channel structure Flashcards
WHat do ion channels look like?
What do they differ by?
- Large polypeptides with 2 or more membrane-spaning subunits (all ion channels are dimer or tetramers), surrounding a pore (aqueous so ions can pass), homomeric or heteromeric
- Differ by ion selectivity + gating mechanism
Potassium channels (4)
Who + structure + gated by + mechanism
- 1998: Roderick MacKinnon first solved the structure of an ion channel with KcsA from Streptomyces Lividans (bacteria in soil)
- Homotetramer (made of 4 of the same subunits)
- Not VG but PH gated
- When it opens, a bunch of ions will flow into the inner chamber but selectivity filter make sure only K+ goes through.
KcsA (3)
selectivity filter + structure/subunits + p-region
- Selectivity filter there are 4 potential binding sites that can bind potassium but only 2 happen at a time due to electrostatic repulsion. You cannot cram that many + charge in a small space.
- Made of 2 transmembrane subunits and a loop helix loop on the extracellular region known as the P-region or p-loop. You need 4 of those for a channel. So 8 transmembrane domain in 1 KCSA channel.
- P region has the pore helix that contributes to shape of pore.
KCSA vs Shaker
KCSA is prokaryotes and shaker is eukaryotic. Shaker is VG but highly resembles each other at the selectivity filter.
Ions in solution are surrounded by ——–.
Selectivity is not based solely on ion diameter explain this:
- waters of hydration after the ionic bond breaks
- The K+ crystal radius is 0.133nm and Na+ is 0.095nm. However, sodium is smaller so the positive charges is more concentrated in a tighter space and holds onto water tighter. This attracts more H2O and acts as a bigger ion then K+ and has a bigger H2O shell and less likely to lose H2O.
Selectivity filter (3)
What + what determines selectivity + Need to be what to fit thru?
- Narrow regions in ion channel pores that act as molecular sieves
- Pore size and chemical interactions with residues lining the pore determine selectivity
- Need to be partially or compltely dehydrated to fit through
Sodium selectivity pore:
Na+ is partially dehydrated to fit through. K+ is too big to fit through.
K+ selectivity sequence (2)
What + highly….
- Thr- X- Gly- Tyr- Gly
- Highly conserved from prokaryotes and eukaryotes
K+ Selectivity how does it work:
Potassium selectivity in K⁺ channels is achieved through a narrow selectivity filter lined with carbonyl oxygen atoms from the protein backbone. These oxygen atoms create a cage-like coordination environment that mimics the hydration shell of K⁺, allowing the ion to be stabilized after dehydration (O is electronegative functional group and has - charge to attaract K+). The filter consists of four subunits arranged in a square, with oxygen atoms positioned at each vertex to precisely fit K⁺ but not smaller ions like Na⁺, which cannot form optimal interactions. This precise geometric and electrostatic arrangement ensures that only K⁺ ions can pass through efficiently.
What propels K+ through the SF and how are they arranged in the SF?
Propelling through is concentration gradient and repulsion between K+ ions.
K+ can be at either 4 site but never seen irl becayse the + charge repel eachother so water molecule is inbetween.
Selectivity filter is at the —- not —–.
- at the exit not entrance (extracellular side)
K+ must be —- to fit through the filter before being ——
- dehydrated
- rehydrated
Vestibule
Inner chamber of channel
Most VG channels form —– shape so transmembrane segment cross at the bottom when close and when activation gate open they ——.
- Inverted cone
- tilt slightly
Potassium channel selectivity filters are remakably percise and 10K times more selective for K+ then Na+. Why?
- All because of the structure arrangement of the carbonyl protein backbone at the filter. Carbonyl groups lining the selectivity filter form favourable electrostatic interactions with K+ but are too widely spaced to interact effectively with Na+. Na+ thus favour their shell of water and do not pass through filter due to being too big.
There are 3 major family of K+ channel:
- 2TM K Channels
- 4TM K channels
- 6TM K Channels
The 2TM K+ channels (5)
subunits + aka/bc +structure subunits that make it up + pore forming region contains + block
- 2 transmembrane helices per subunit(simplest)
- Inward rectifiers: Pass current more easily in the inward direction then out (pass K+ into cell)
- Homotetramers (4 of the same subunit making 8 transmembrane subunit for 1 2TM channel)
- Pore forming region contains pore helix and K+ selectivity sequence
- There is a voltage dependent block of channel pore by Mg2+ and polyamines when membrane is depolarized. If you hyperpolarize the cell, you pull out the + cation and relieve the block.
2TM channels conduct more current when (2):
potential/range +how
When the membrane is hyperpolarized (more negative inside), the channel opens, allowing K⁺ to flow. At membrane potentials more positive than -40 mV, the channel remains shut. These channels only conduct when the voltage steps in the negative direction (hyperpolarizing), meaning the electrical gradient can overshoot the chemical gradient, further hyperpolarizing the cell. Since the inside of the cell is more negative, it pulls K⁺ ions inward against their usual outward concentration gradient.
4TM K+ channel (4)
AKA + subunit + structure/subunits that make up it + P-region
- AKA leak channels which are always open K2P
- 4 transmembrane domains and 2 pore regions per subunit
- Homo or heterodimers (2 subunit form a complete channel and so a 4TM channel has 8 transmembrane segment)
- 2 extracelullar loop that makes up P-region
4TM channels current + IV curve (2):
- Pass currents in both direction because they are open at all voltage
- The IV curve has an outward current (K+ is leaving cell) at voltages above -90mV aka Eq potential of K+. At lower voltage then that, K+ influx.
Membrane is leaky to K+ means interior negative as K+ take positive away
6TM K+ channels (7)
AKA + subunit + structure/subunits that make up it + # of channel/subfamilies in humans + T1 + Beta + voltage sensor
- AKA Kv channels/ Voltage gated
- 6 transmembrane segments labelled S1-6. Hairpin between S5 and S6 known as H5 or P (selectivity filter)
- Homo or heterotetramers (4 subunit = 24 transmembrane in VG-K+ channels)
- 40 channels, 12 subfamilies in humans
- The T1 (tetramerization domain): Match up subunit to make homo/heterotetramers
- B: Not all K+ has B subunit (modify function)
- S4 is the voltage sensor and active at depolarization voltages
6TM K⁺ channels have six transmembrane (TM) segments per subunit. These channels typically include:
volatge sensing domain + pore forming
S1-S4: Form the voltage-sensing domain (VSD), with S4 containing positively charged residues that detect changes in membrane voltage.
S5-S6: Form the pore domain, with the selectivity filter between them allowing K⁺ selectivity.
K+ VG channel types (2):
- A-types
- Delayed rectifiers (majority): Repolarizes AP
6TM K+ channels are voltage sensitive and activated at ———. They regulate ———-. There are 2 type, explain what they do:
- depolarized voltages (+ to -50mv) and close at negative potentials.
- shape and firing pattern of AP
- Fast KA vs classical delayed (KVdr) channels. KVdr is important to AP repolarization (recovery phase) and take longer to open, current is sustanined overtime. KA is a fast current rise and shuts off quick.
which channel is sensitive to TEA and known as outward rectifier?
KVdr/delayed rectifiers
6MT
What type of residues are present every third position in the S4 voltage sensor?
Positively charged residues (Arginine or Lysine).
6MT
What is the repeating amino acid pattern in the S4 voltage sensor?
Arg/Lys – X – X – Arg/Lys (where X is a neutral residue).
6MT
Why is the S4 segment considered hydrophilic?
Because it has positively charged residues every third position, making it unfavorable for embedding in the nonpolar membrane.
6MT
Why is the S4 segment pulled toward the intracellular side at resting potential (-70mv)?
Because the inside of the membrane is more negative, attracting the positively charged residues of S4.
6MT
What happens to the S4 segment in close and open state?
The sliding Helix model
The paddle model
The transporter model
Comparision of the 3 models of S4 translocation:
Support?
- Sliding helix/helical screw: Water crevice S4 is sliding in. Perdicts a large translocation measured by gating current that suggests big movements. Movement tugs on S5 linker to open.
- Paddle model: Supporter by charges embedded in bilayer. Pull on S4-S5 linker to open actviation gate.
- Transporter model: Smaller tilt with little to no translocation.
What was the support for sliding helix?
What is a common feature for all 3 VG sensing theory (2)?
- S4 involved
- Charges move inside to outside for everymodel
VG K+ channel has 2 states:
- Open at -50mV or above
- Closed at rest or -70mV
Na+ has 3 main states and 2 gates:
- Closed at resting (-70mV). The activation gate on the bottom is closed, inactivation is open.
- Open at >=50mV.
- Inactivated at +30mV.
Subunit structure of Nav Channels
Subunit structure of Nav Channels (2)
- 9 pore forming subunits in mammals (Nav 1.1-1.9) Large alpha subunits
- 2 auxillary subunits B1-B4 (4 different beta subunit/gene that can occur) that modulate channel function, expression and trafficking (ex: time to activate, how long to close). Lies towards extracellular side with glycosolated carbohydrate chains.
Alpha subunit structure of NaV channels (3)
alpha + S4 + p-loop
- 4 homologous repeats of 6TM domains (monomer) structurally laid out the same way. 1 alpha subunit is made up pf 24 TM segments in total.
- S4 is voltage sensing domain linked to pore domain via S4-S5 linker. As S4 move across membrane, it would tug on S5/S6 and open/close the pore. Every third AA is + in S4 and in between is hydrophobic AA
- S5-S6 has P-loop (H5) with 2 helices
Na+ selectivity sequence (3)
motif + organization + why certain AA
- DEKA motif (Asp-Glu-Lys-Ala) within selectivity filter of pore domain that forms a ring at constriction site. This is the key sequence that needs to be there to select for Na+ and not K+.
- 1 AA in each domain of alpha subunit in S5-S6 linker (H5)
- Asp and Glu is negative, Lys is positive and Ala is nonpolar. It is hypothesized that electrostatic repulsion from Lys places Na+ ion (partially dehydrated) next to negative residues. If you remove lysine you lose Na+ selectivity.
L3 (third intracellular loop) (2)
Connects what + mechanism of action
- Connects S6 (DIII) to S1 (DIV)
- Fast inactivation particle IFM motif (isoleucine, phenylalanine, methionine). IFM is triplets of AA resideues that swings into activation pore to block it because hydrophobic pocket is exposed when channel is in activated state. This blocks the pore, preventing further Na⁺ influx, which is essential for proper neuronal signaling. Because the channel take a while to go back to recovery/close structure, you dont want Na+ constantly flowing through channels and cause hyperexcitability in brain.
What are the two features that are different between K+ and Na+ channels?
- Alpha subunit divided into 4 domain with 6TM segments per domain.
- P loop (H5) has 2 helices
Describe the 3D structure of a cryo-EM structure of human NaV1.4:
- Domain 1-4 are all seperated around all the S5/S6 of all the 4 domains which combine and cross to form the center.
Gating of NaV channels: Negative potentials (Vrest) (3)
S4 position/organization+ S5/S6
- All voltage sensors S4 in downwards position. Positive charge is closer to the intracellular side of the neuron.
- Pore forming in center (S5/S6)
- 4 voltage sensing domains 1 per structural domain
Gating of NaV channels: Activation (Membrane depolarization) (3)
Gating charges + S4 + activation gate
- Move gating charge outwards (helical screw)
- S4 of voltage sensor DIV lags behind and remains downwards.
- Opens activation gate which is at intracellular side of VG-Na+ channel so Na+ can flow in and cause further depolarization (responsible for rising phase of AP)
Gating of NaV channels: Inactivation (Peak depolarization) (3)
mV+ mechanism of action + restoration to rest
- Around +30mV of AP
- Outward of S4 (DIV) so all S4 in the 4 domains are up. This exposes the receptor site for IFM inactivation particle.
- Cell start to repolarize due to VG-K+ channel which brings voltage sensor S4 down
Why is activation not possible during K+ efflux of absolute refractory but possible during relative refractory?
- During absolute refractory, Na+ channels are already open (no further effect) and VG-Na+ channel are inactivated (no Na+ current)
- During relative refractory, it is possible for another AP as VG-Na+ back at rest state (inactivation gate has opened, IFM fallen away from hydrophobic receptor site, S6 crossed) but you need stronger stimulus because it is further from threshold.
