Introduction to Neural Signaling
Ion channels are membrane proteins which allow the follow of normally impermeant ions across the hydrophobic "sea" of cell or organelle membranes. The channel protein can be constitutively open or can be "gated" open (or closed) by signals that affect the proteins conformation. The signals can be ligands (in the form of hormones or neurotransmitters), post-translational modifications (mostly phosphorylation, dephosphorylation), or more difficult to understand events (change in transmembrane potential, pressure, or temperature). Gated channels are perhaps studied through through role in neural signaling.
Neurochemistry is one of the most explosive areas of biological research. Scientists are now starting to unravel the molecular bases for memory, cognition, emotion, and behavior. The next decades will bring truly revolutionary understanding of brain chemistry and along with it the potential to alter human mood, memory, and to treat mental illness such as schizophrenia much more effectively. The human brain, with about 86 billion neurons. Compare this with some estimates for the number of stars in the Milky Way galaxy (about 100 billion, a number derived from luminosity and mass measurements). Now imagine that each neuron can form connections - synapses - with 1000 to 10,000 other neurons. Throw in another 86 billion brain glial cells and you have one of the most complex structures in the universe. This section will explore the biology and chemistry of neurons.
We will discuss two kinds of neurons - those that interact with muscles at the neuromuscular junction and those that interact with other neurons in the central nervous system. Neurons consist of a single, nucleated cell body with multiple signal-receiving outgrowths (dendrites) and multiple-signal sending outgrowths (axons) which end in a terminal button. These interact through the synapse with dendrites on other neurons.
A presynaptic neuron can stimulate an adjacent postsynaptic neuron by releasing a neurotransmitter into the synapse between the cells, which binds to a receptor in the membrane of the post-synapatic cell, stimulating the cell.
We will discuss the events which cause the post-synaptic cell to "fire", but we will not discuss the immediate events which lead to the release of neurotransmitter by the presynaptic neuron.
Neurons (as do all cells) have a transmembrane potential across the membrane. Transient changes in the membrane potential are associated with neuron activation or inhibition. This arises in part due to the imbalance of ions across the membrane which was established by the Na+-K+-ATPase in the membrane. This electrogenic antiporter transfers 3 Na ions out of the cytoplasm for every 2 K ions it transports in. Likewise Cl- has a much higher level outside the cell. Membrane potentials are determined not only by the size of the ion gradients across the membrane, but also the differential permeability of membranes to ions. As we saw previously, synthetic bilayer membranes are not very permeable to ions .
|sodium||<1.6 x 10-13 (lowest)|
|potassium||<9 x 10-13|
|chloride||<1.5 x 10-11 (highest)|
Sodium would be expected to have a lower permeability than potassium since it has a higher charge density. It also is largest in effective size due to the larger hydration sphere around the ion arising from its higher charge density. Chloride would be expected to have the highest permeability since it has the lowest charge density (due to repulsion of the electron cloud in the negative ion). Intracellular charged proteins (which are mostly negative) are not permeable and help in creating the negative charge imbalance across the membrane.
Much work has been done on the giant axon of the squid, which has uniquely high intracellular potassium (400 mM compared to 20 mM outside) and high extracellular fluid sodium (440 mM vs, 50 mM inside) and chloride (560 mM vs 52 mM inside). Mammalian cell concentrations are much lower, but the relative size of the gradient is about the same.
|Ion||Cell (mM)||Blood (mM)||Permeability (cm/s)|
|potassium||140||5||5 x 10-7|
|chloride||4||110||1 x 10-8|
|X- (neg. macromol.)||138||9||0|
How can we account for the markedly greater permeabilities of ions (1000x to 1,000,000 x) in mammalian cell membranes compared to synthetic lipid vesicles? Previously, we showed that glucose has a greater permeability through red blood cell membranes than through synthetic liposomes because of a membrane receptor that allows facilitated diffusion across the membrane and down a concentration gradient. The same thing is true of ion permeabilites in intact biologicial membranes. These membranes have several types of selective ion channels (nongated - always open, and gated - open only after specific conformational changes). The nongated channels dramatically increase the permeability of membranes to ions, as the glucose transport protein increased the permeabilty to glucose. It turns out that this differential permeability contributes to the transmembrane potential. Ion channels in nerve and muscle can move ions across the membrane at a rate up to 109/s, which is comparable to kcat for the best enzymes.
If we envision channels as pores, how can we account for the selectivity of the channel to specific ions. A larger pore should admit any ion less than a maximal size for the pore, so it is hard to image the nature of the selectivity filter. Because of this, many people discounted the ideas of channels in favor of a transporter, which would bind the ion selectivity and then, through conformational changes, move the ion across (much like the Na/K ATPase we discussed in the previous guide). This model could not, however, account for the incredible rates of ion flux across the membrane. Selectivity can be accounted for by a channel that contains a narrow opening that acts as an ion sieve. The ion looses most of its hydration sphere and would form specific interactions with amino acid side chains in the pore region. Such an interaction would be transient and not too tight since the ion must pass through the membrane. As we will see later, these ion channels:
- pass ions down a concentration gradient in a thermodynamically favorable process
- are specific for certain ions (although a few are less selective and will pass Na, K, Ca, and Mg ions)
- allow ion flow through either ungated or gated channels.,
- saturate with increasing ion concentration (even though as concentration increases, the ions have a greater thermodynamic drive to pass through the channel). This is consistent with binding of the ion at a selectivity filter in the narrow part of the pore. The KD for the interaction is usually in the mM range and indicates weak binding with large dissociation rate constants (koff).
Several questions arise about the distribution of ions and the magnitude of the transmembrane potential.
- How are the ion gradients established?
- How does the transmembrane ion distribution contribute to the membrane potential?
- How can the resting electrochemical potential and the ion distribution be maintained?
The answer to these questions will be illustrated using studies on two types of brain cells, glial cells (which function as protectors, scavengers, and feeder for brain neurons) and neurons. Both types of cells have transmembrane potentials.
1. The transmembrane ion gradients for ions can be established by different mechanisms. One uses ion-specific ATPases (P-type ion transporters), such as we discussed with the Na/K ATPase. This transporter ejects 3 sodium ions from the inside of the cell for every 2 potassium ions it transports in, all against a concentration gradient. Since it is an electrogenic antiporter, it helps generate the potential. Additionally, specific ion channels also contribute (as described below) to the transmembrane gradients and potentials.
2. The harder question is how the ion distribution contribute to the membrane potential. Two things must occur for a membrane potential to exist: First, there must be a concentration gradient of charged ions (for example, sodium, potassium, or chloride) across the membrane. Second, the membrane must be differentially permeable to different ions. If the membrane were completely impermeable to ions, then no movement of ions across the membrane could occur, and no membrane potential would arise. If, however, membranes are differentially permeable to the ions, an electrical potential across the membrane can arise. Remember, synthetic bilayers are quite impermeable to ions, given the hydrophobicity of the internal part of the bilayer. Likewise it is quite impermeable to glucose. It turn out that glial cells appear to have only a non-gated potassium channel, which allows the outward flow of potassium ions down the concentration gradient. The inside will then have a net negative charge since impermeable anions remain. The chemical potential gradient causes this outward flow of potassium ions. As more ions leave, the inside gets more negative, and a transmembrane potential develops which resists further efflux of potassium. Eventually they balance, and the net efflux of potassium stops. The resting transmembrane potential reaches -75 mV which is exactly the value obtained from the equations we will derive below. Since glial cells appear to only express a nongated potassium channel, their resting potential is equal to the potassium equilibrium potential.
(Note: above figure has mistake. Cl- ion , assuming that is not permeable, will not change across the membrane at equilibrium.)
Neurotransmitter Activation of Neurons
What happens when a neurotransmitter binds to a receptor on the post-synaptic cell? We will study two examples. The first is the simplest: binding of the neurotransmitter acetylcholine, released by a motor neuron, to its receptor on muscle. This region is called the neuromuscular junction. Binding of acetylcholine will lead to a transient depolarization of the muscle cell. Next we will discuss the interaction of a neurotransmitter with a post-synaptic neuron in the central nervous system. This is a much more complex system. Their differences are described below:
In neurons interacting with muscles:
- Most muscle fibers are innervated by only one neuron - a motor neuron
- Neurotransmitter release at the neuromuscular junction leads only to muscle excitation, not inhibition.
- All fibers are excited by the same neurotransmitter - acetylcholine.
In the central nervous system, life is more complicated:
- Stimuli are received from hundreds to thousands of different neurons.
- Nerves receive both excitatory and inhibitory stimuli from neurotransmitters
- Different kinds of receptors are present to receive stimuli, which control the activity of different kinds of channels.
- The ion channels in neurons are gated by a variety of mechanisms in addition to changes in membrane potential, including gating by heat, cold, stretch, or covalent modification.
- Most nerve cells have a resting potential of about -65 mV compared to a -90 mV for a muscle cell.
What happens when a neurotransmitter binds to the receptor on the post-synaptic cell? A depolarization occurs (mediated by conformational changes in the transmitter-receptor complex), raising the membrane potential from the resting equilibrium level. What happens next depends on the identity of the post synaptic cell. In the muscle cell, the rising potential caused by binding of acetylcholine ultimately leads to muscle contraction by opening intracellular organelle membrane calcium channels. In a neuron, the rising potential triggers an action potential by opening voltage-gated sodium channels. The potential rises to about + 35 mV, but does not reach the Na ion equilibrium potential, because the high positive potential opens a voltage-gated potassium channel. The potential then falls until it reaches the K ion equilibrium potential where the cells is hyperpolarized. It slowly then relaxes back to the resting potential of -60 mV. This wave of changes in potential sweeps down the post-synaptic cell membrane and is the basis for the "firing" of the neuron.
Here is an animation of a neuron firing showing all the key players. (produced by PhET, University of Colorado, Boulder).
Proteins of the Neural Synapse
We must now account for the rise and fall of the membrane potential to a variety of neurotransmitters, including the cholineric transmitters (ex. acetylcholine), catecholamines (dopamine, epinephrine, norepineprine), amino acid derivatives (ex. Glu, Asp, N-methyl-D-Asp, Gly, gamma-amino-butyric acid -GABA), and peptides (endorphins, encephalins). We will consider five membrane proteins:
Figure: five membrane proteins
- Na+-K+-ATPase: We discussed this in the previous section on active transport. It transports both sodium and potassium ions against a concentration gradient using ATP as an energy source. The protein is a sodium dependent ATPase. Without this protein, the membrane potential could not be maintained since the sodium and potassium gradient would collapse. It also contributes to the potential since its is an electrogenic antiporter. (In addition, we have seen that ungated potassium and sodium channels are also present.)
- Neurotransmitter receptor: The receptors we will consider here are typically ligand-gated ion channels. Once ligand binds, a conformational change occurs in the protein, allowing a flow of ions down a concentration gradient. Depending on the nature of the ion, the channel either initiates depolarization (when Na+ enters from the outside and raises ΔΨ) or inhibits depolarization (when Cl- enters from the outside and lowers ΔΨ). When chloride channels open, they hyperpolarize the transmembrane potential. Stimulatory neurotransmitters (like glutamate) lead to depolarization of the membrane, while inhibitory neurotransmitters (like gamma-aminobutyric acid) lead to hyperpolarization of the membrane (make the potential more negative).
- Na+ channel (voltage-gated): When the ligand-gated channel depolarizes the membrane to some threshold value, sodium channels undergo a conformational change and open allowing Na+ ions to flood into the cell, raising the potential to a positive approx. 33 mV (a value lower than the the equilibrium sodium potential). This membrane protein is a voltage-gated channel, not a ligand gated one. Somehow, it senses a change in the transmembrane potential initiated by opening of the ligand-gated channel.
- K+ channel (voltage gated): When the membrane potential reaches around +25 mV or so, the K+ channel, a voltage-gated membrane protein, alters its conformation, allowing K+ efflux from the cells, lowering the potential until it reaches the potassium equilibrium potential. It slowly relaxes back to the cell resting potential of about -60 mV. Recently, the crystal structure of the K+ channel from Streptomyces lividans was determined (Science, 280, 69-77, 1998): "All K+ channels show a selectivity sequence of K+, Rb+ > Cs+, whereas permeability for the smallest alkali metal ions Na+ and Li+ is immeasurably low. Potassium is at least 10,000 times more permeant than Na+, a feature that is essential to the function of K+ channels.....Two aspects of ion conduction by K+ channels have tantalized biophysicists for the past quarter century. First, what is the chemical basis of the impressive fidelity with which the channel distinguishes between K+ and Na+ ions, which are featureless spheres of Pauling radius 1.33 ï¿½ and 0.95 ï¿½, respectively? Second, how can K+ channels be so highly selective and at the same time, apparently paradoxically, exhibit a throughput rate approaching the diffusion limit? The 104 margin by which K+ is selected over Na+ implies strong energetic interactions between K+ ions and the pore. And yet strong energetic interactions seem incongruent with throughput rates up to 108 ions per second."
- Cl- channel: If these channels (typically ligand-gated) are open, they will hyperpolarize the cell and make it more difficult to fire.
Voltage-Gated Sodium Channel
Patch clamp recordings of the sodium channel reconstituted in vesicles shows it to be a voltage-gated channel. Two potent neurotoxins, tetrodotoxin (from Puffer fish) and saxitoxin bind to the channel and act as antagonist (inhibits the activity of the receptor by blocking sodium influx).
The guanidino group of the tetrodotoxin appears to bind with high affinity to the entrance of the channel that interacts with the hydrated sodium ion. Affinity chromatography using tetrodoxin beads have been used to purify the protein. The protein sequence consists of 4 repeating units, each containing 6 transmembrane helices. Ion selectively of the sodium channel is determined by the size of the the ion when it is hydrated by one water molecule.
Hence Li and Na ions can pass through, but not the large K ion. Hydroxylamine and hydrazine both have the same width as a water-Na+ complex, and are both positively charged. These can also pass through the channel. At the narrowest part of the channel a water (and presumably the hydroxylamine and hydrazine) form H-bonds to side chains in the pore while the positive charge on the ion forms transient salt-bridges to either a Glu as Asp in the pore. The open channel can likewise spontaneously inactivate by undergoing another conformational change. Comparison of the sequence of this channel with calcium and potassium channels show extensive sequence and structural similarities. One of the 6 transmembrane helices, S4, appears to be the voltage sensor. Depolarization of the membrane potential rmay result in an outward movement and rotation of the positively charged helixes containing Lys and Arg side chains which had presumably formed salt-bridges with negatively charged side chains within the protein. Depolarization of the membrane results in breaking a few salt bridges, and effectively leads to the movement of 1 or 2 charges on the helix through the membrane. Work occurs when charges are moved through an electric field. Work is also related to the ΔG for the system, which is also dependent on the ratio of the open and closed form of the channel. Other voltage-gated ion channels (for potassium and chloride) have a similar membrane topology and an S4 voltage-sensor helix.
Voltage-Gated Potassium Channel
How can the two essential features of the K+ channel pore - selectivity for the larger potassium ion over the sodium ion, and the near diffusion-limited rate of potassium ion flux through the channel be reconciled?
The selectivity filter is composed of many stacked rings of oxygens that can interact with a dehydrated K ion but not with a dehydrated Na ion which can not approach close enough to form significant interactions. Surrounding the filter are twelve aromatic amino acids which constrain the size of the pore opening. The interactions of the filter O's with the K ion makes up for the energetically disfavored dehydration of the ion. The filter contains two K ions which repel each other, assisting in the vectorial discharge of the ions through the membrane. These ions must form weak interactions with the selectivity filter. The actual pore is mostly hydrophobic, which facilitates flow-through of the ions. The central cavity of the pore can hold some waters molecules in addition to the K ions which helps stabilize the ion in the middle of the pore.
The latest (2005) new structure (Long et al) from MacKinnon's group of the another eukaroytic K+ channel (in its open state) is more consistent with previous expectations. Instead of using antibodies to stabilize the voltage-sensor domains, they crystallized the physiological channel, which in eukaryotes consists not only of 4 identical membrane spanning subunits, but also an addition domain T1 and another protein, beta. The T1 domain consists of the N terminal regions preceding the first membrane-spanning helix, S1, of each of the four monomers of the channel and is located intracellularly at the entrance point of the channel. Four conserved Arg resides on S4, part of the voltage-sensor domains, are hypothesized to move under the influences of forces arising from changes in the membrane's electric field initiated by ion movement through other ion channels in the membrane. Mechanical work is done by the electric field on the voltage sensor as the charged Arg residues are moved through the electric field and the protein conformation is altered. In turn, the S4 and coupled S5 helices of the voltage sensor do mechanical work on the pore by altering its conformation to open/close the pore, specifically at the activation gate of the pore. This seems quite similar to how iron movement into the heme plane in hemoglobin on oxygenation pulls the proximal His on the F8 helix which then transmits a conformation change to other helices in the subunit, leading to cooperative conformational changes in the tightly packed protein. Two of the Arg residue on the S4 sensor are facing the lipid interface while the other two are buried in the voltage-sensor domain. Their work shows that this domain is clearly separate from the pore domain, much as the extracellular ligand-binding domain of neurotransmitter-gated ion channels are separated from the pore domain. In this case, however, the voltage-sensor domain is buried in the membrane.
The main inhibitory neurotransmitters are GABA (gamma-aminobutyric acid, which is made from Glu through decarboxylation of the α-C group) and glycine. They bind to transmitter-gated chloride channels, which when open hyperpolarize (make more negative) the transmembrane potential. Benzodiazepines (like Valium and Librium - anti-anxiety and muscle-relaxing agents) and barbituates (like phenobarbital-hypnotics) bind at allosteric sites on the GABA receptor and potentiate the binding of each other and GABA. This receptor is also affected by alcohols and anesthetics. Let's focus on the GABAA channels since it binds so many interesting pharmaceutical drugs.
The structure below show the receptor
The structure of the GABAA receptor bound to GABA and picrotoxin is shown below.
A view showing just bound picrotoxin is shown below. (File modified by HVJ and uploaded to LibreTexts)
Try this: iCn3D display: GABA bound between the A and B subunits (6huj)
Here is bound gaba in 6huj