W5L3 Flashcards
Molecular structure of nicotinic AChR
Pentameric (5 subunits) - 2 alpha, 1 beta, 1 gamma, 1 delta
It is acetylcholine receptor
Acetylcholine binding site are on the 2 alpha subunits
- does not require 2 Ach in order to open the channel
Each of the 5 subunits have the same 4 transmembrane domains
- TM1, TM2, TM3, TM4
- ends of the transmembrane (that is continuous) has NH2 and COOH
TM2 of each subunit lines the pore of the channel
Selectivity filter and gating of nAChR
Negatively charged residues on M2 (lines the pore, the inner part of the channel) forms a selectivity filter for a
cation channel
- repels anions away from getting into the channel
- channel is only permeable to cations (no selectivity for Na+, K+, Ca2+, etc); it is non-selective cation channel; only permeable to cations
Voltage-gated K+ channels are tetramers
4 subunits assemble to form the functional channel; homo (all subunits the same) tetramer
Each subunit is comprised of 6 membrane spanning domains (S1-S6), with cytoplasmic amino- and carboxy-terminal regions.
- the pore is bw S5 and S6
Some structural features of K+ channels
The S4 segment is full of charges and represents the voltage-sensor region
- senses the membrane potential
- drives the channel either open or closed
The pore loop contains the selectivity filter, and is also the site for binding of various blockers
- pore loop is between S5 and S6
- channel blocker: TEA= tetra ethyl ammonium
If you remove water from K+ ions, they need energy
Structure of a K+ channel voltage sensors
MacKinnon & colleagues found that K+ channel voltage sensors resemble charged ‘paddles’
- cell exterior is positive, interior of cell is negative; like the resting membrane potential
S4 voltage sensing regions contain + (pos) charged arginines
A hinged movement gates the channel pore open or close, allowing K+ to exit cell or not
Voltage-gated Ca2+ and Na+ channels have 4 domains
It is MONOMER; one single subunit needed
- but the subunit is HUGE
Ca2+ or Na+ channels: only one subunit to form the pore and this subunit is made of 4 domains (I – IV), each of which has the 6 membrane spanning domains.
One Na+ subunit can form a functional channel
Has voltage sensor and pore
Has voltage-dependent inactivation bw the third and the fourth domain
Some channels have activation and inactivation gates
CLOSED
- activation gate is closed
- inactivation gate is open
OPEN
- both activation and inactivation gate are open
INACTIVATED
- activation gate open
- inactivation gate closed
only some channels are like this
N-type inactivation of a K+ channel(A-type K+ channel)
For this channel, the inactivation depends on amino terminus
- Remove 20 amino acids at the N-terminus : no inactivation
- Restore 20- amino acid peptide: restores inactivation
Ball and chain model of channel inactivation
Depolarization leads to channel activation, followed by a delay with blocking of the pore by the N-terminus (inactivation state)
Membrane hyper-polarization is then required for the removal of inactivation state
Gap Junctions
Type of membrane channel
Gap junctions are a special class of membrane channels that couple neighbouring cells together by creating pores from the cytoplasm of one cell to the cytoplasm of another.
Aqueous pores
Hexameric proteins aligned in neighboring cells
Inside of one cell to the inside of another cell; direct
Permeable to ions and small molecules
Molecular structure of gap junction channels
Each molecule is called a connexin
6 connexins assemble to form a hemichannel
2 hemichannels from neighboring cells dock together to form a functional gap junction channel.
Can be:
1. Homomeric homotypic
- each hemichannel is made of the same connexin
- both hemichannels are identical
- Homomeric heterotypic
- each hemichannel is made of the same connexin
- but the hemichannels are different between the 2 cells - Heteromeric Heterotypic
- each hemichannel is made of NOT the same connexin (any combo)
- and the hemichannels are different between the 2 cells
Properties of gap junctions
1) Provide cytoplasmic continuity between cells, so current passes between cells, typically in both directions (electrical synapse)
2) An electrical synapse has a very short synaptic delay (~ 0.1 ms)
3) Synchronous activation of a large number of cells in heart and smooth muscles.
4) Channels are large enough to pass small molecules ( < 1kD) between cells, e.g., nutrients, metabolites and signaling molecules.
5) Increased intracellular acidity (decreased pH) or Ca2+ close gap junctions
Why do we need patch clamp?
Monitoring cell electrical activities.
Recording under controlled conditions.
1 micrometer, not that sharp but still sharp
Needs low resistance
Patch clamp
- Glass pipette approaches a cell, controlled by a manipulator
- Once you get close to the cell, you can monitor the resistance. can form a giga seal to monitor resistance change. Giga seal means very little ions leave the cell
- Then it forms a strong suction to rupture cell membrane. Then it opens up the part of the membrane, allowing you to have whole cell recording
