Structure and molecular biology of ion channels Flashcards
What is the plasma membrane made of ?
Which compounds can/cannot cross it ?
How does it control movement across it ? - why is this important ?
The plasma membrane is composed of a lipid bilayer and associated integral and peripheral proteins.
Most biological molecules (except for ex. steroid hormones) are hydrophilic and cannot cross directly
through the hydrophobic bilayer. Their movement is controlled by integral membrane proteins such as
channels, pumps and transporters. The plasma membrane constitutes therefore a selective barrier between the inside and outside of the cell. This allows the cell to optimise the internal environment for certain biological functions and to create rapid specific changes in that environment as a way of responding to events in the outside world. Cells can thereby generate
signals.
How are ions in solution ?
How does this affect the probability of this ion crossing the lipid bilayer ?
Ions in solution are surrounded by a cloud of water molecules (hydration shell) that are attracted by the net charge of the ion.
It is extremely energetically unfavourable, and therefore improbable, for the ion to leave this polar environment to enter the non-polar environment of the lipid bilayer.
What are ion channels ?
Ion channels are large integral membrane proteins that form aqueous pores through the plasma membrane that allow ions to cross. Ions always flow down their electrochemical gradient through channels (passive transport).
The basic unit of signalling in the NS is the AP.
What are the 3 essential features of axonal information transfer ?
- There is a threshold for generation of action potentials that guarantees that small random variations in the membrane potential are not misinterpreted as meaningful information;
- The all-or-none law guarantees that once an action potential is generated, it is always full size, minimising the possibility that information will be lost along the way;
- The strength-latency relationship and the refractory period, together with the threshold, allow the encoding of information in the form of a frequency code.
What did crucial experiments in the ’50s by Cole, Curtis, Hodgkin, Huxley and Katz reveal ?
During the action potential the conductance of the membrane to ions increases.
The depolarisation that initiates an action potential causes a transient change in the membrane that briefly switches its predominant permeability from K+ to Na+.
Subsequent isotopic K+ flux-ratio experiments of Hodgkin and Keynes led them to formulate the following
hypothesis: “(…) Ions cross the membrane along a chain of negative charges or through narrow tubes or channels… in which they are constrained to move in single file with several ions in the channel at any moment.”
What was Armstrong and Hille’s working hypothesis in the late 1990 concerning the movement of ion across lipid membranes ?
Ions are passing through aqueous pores that they called
channels
They have swinging gates that open and close them in
response to voltage changes
We can study their architecture by using electric currents to measure gating, permeation and block
Channel blockers are molecules that enter the pores and physically plug them
How can the “on channel theory” be experimentally tested ?
Three major technical approaches leading to breakthrough discoveries:
The patch-clamp technique and single channel measurements;
Molecular cloning of voltage-gated ion channels and structure-function studies;
Crystallisation and high resolution 3D structure of ion channels.
Who discovered patch clamp ?
What does it allow ?
The patch clamp technique developed by Neher and Sakmann allows to measure the tiny ionic currents flowing through single channels.
In other words, it allows to measure the activity of a single protein molecule!
What are the 5 main functional features of voltage-gated ion channels ?
- Voltage-gated ion channels are water-filled pores crossing the membrane and opening or closing in
response to changes in the membrane potential; - The flux of ions through these ion channels is
passive, requiring no expenditure of metabolic energy.
The direction and eventual equilibrium for this flux is
determined not by the channel itself, but by the
electrochemical driving force across the membrane; - Ion channels select the types of ions that they allow
to cross the membrane (selectivity filter). They discriminate among possible permeant ions on the basis
of ionic charge, ionic diameter and weak electrostatic
interaction of the ion with charged amino-acid residues
that line the wall of the channel; - The transition of the channel between closed and
opened states is called gating. Gating mechanisms
involve conformational changes in channel structure.
The “gate” of voltage-gated channels responds to changes in the membrane potential; - Many, but not all, voltage-gated ion channels can enter a refractory state following activation. This process is called inactivation.
Which technique allowed the following question to be addressed ?
Are there proteins functioning as ion channels, complete of all the functional features revealed by
electrophysiological experiments?
What are their structural characteristics?
Do the different functional features (for ex. voltage sensing, selectivity filter, gating, inactivation) have structural correlates?
These questions could be addressed after the
cloning of the first voltage-gated ion channel by the group of Numa.
What can the primary sequence of a channel protein tell us about it’s structure ?
The hydropathy plot was obtained by assigning to each amino acid in the protein a hydrophobicity/hydrophilicity value, corresponding to its ability to interact
with water:
non-polar = hydrophobic»_space;> +
polar = hydrophilic»_space;> -
A running average over several amino acids (optimal window to detect membrane-spanning stretches = 19 aa) is calculated around each amino acid in the sequence and plotted against its position along the protein chain.
Hydrophobic stretches of amino acids of sufficient length are likely to be in direct contact with the lipid membrane. Conversely, hydrophilic regions of the protein are more likely to be exposed to the extracellular or intracellular aqueous milieu.
How can one experimentally test whether a cloned protein is an ion channel ?
Expression in heterologous systems and electrophysiological characterization :
- expression of channels in Xenopus oocytes
- transfection of clonal cell lines and site-directed mutagenesis
What essential domain do VGICs possess that allow them to respond to voltage ?
A voltage sensor, usually the S4 transmembrane domain, containing a positive amino acid (Lys or Arg) every 3rd amino acid
Which electronic device did Roderick Mackinnon compare ion channels to ?
Because of their voltage sensor, ion channels can act as transistors. Based on its crystal structure, Roderick MacKinnon and coll. have proposed a new way the
voltage sensor moves across the membrane to open potassium channels.
What do the latest discoveries propose concerning the movement of the voltage sensor of VGICs upon depolarization ?
This latest models is based on voltage-clamp fluorometry and molecular dynamics simulation and suggest a molecular mechanism of activation in which the primary voltage sensors, S4s, rotate by ~180 ̊ as they move “outward”. A subsequent tilting motion of the S4s and the pore domain helices, S5s, of all four subunits induces a concerted movement of the channel’s S4-S5 linkers and S6 helices, allowing ion conduction.
