5.3: Properties of vesicles

Types of vesicles

Molecules of neurotransmitters are often stored in synaptic vesicles before being released. Synaptic vesicles are tiny spheres of lipids just like the cell membrane. These vesicles can be roughly characterized into one of two classes:

1. Small vesicles. These vesicles have a diameter of 40 nanometers and a volume of about 30 cubic microns. Given the size of neurotransmitters, we can estimate at somewhere on the order of thousands to tens of thousands of molecules of neurotransmitter can be stored in each vesicle. Small vesicles store most of the neurotransmitters we most often think of, including glutamate, GABA, dopamine, and norepinephrine, for example. Small vesicles are almost always exclusive found in the axon terminals.

2. Large dense-core vesicles. These vesicles are much larger than small vesicles, with a range of diameter from 100 to 250 nanometers. They store peptides such as dynorphin or enkephalin, which have chemical structures much larger than the other neurotransmitters. Since these peptides are packaged into their vesicles near the nucleus, large dense-core vesicles can be found in the cell bodies and all along the axons in addition to the axon terminal.

Loading of vesicles

Vesicles need to be filled with molecules of neurotransmitter before release into the synapse. In small vesicles, filling is only made possible through the action of giant transmembrane proteins called vesicular transporters. These are protein complexes that span the vesicular membranes, with one side facing the intracellular space and other facing the inside of the vesicle. Their main function is to take molecules of neurotransmitter from the intracellular space of the axon terminal and pump them into vesicles.

Electron microscopy

Electron microscopy is a technique that allows for the imaging of subcellular structures that are on the order of nanometers, such as synapses, small vesicles, and large dense core vesicles.

Figure 5.5 Electron microscope image showing small vesicles (cyan) and large dense core vesicles (magenta).

Many of the vesicular transporters are named based on the neurotransmitters that they are capable of recognizing and transporting. Some have a single substrate, such as vesicular GABA transporters (VGAT) which move GABA, vesicular glutamate transporters (VGluT) which move glutamate, and vesicular acetylcholine transporter (VAChT) which moves acetylcholine into vesicles. Others recognize a broad class of neurotransmitters, such as the vesicular monoamine transporters (VMATs), which are responsible for moving monoamines such as dopamine and serotonin into the vesicles.

Vesicular transporters are able to function because the interior of the vesicle is highly acidic compared to the interior of the cell. Vesicles have a high concentration of H+ ions (protons) because of the action of the transmembrane enzyme vesicular ATP-ase, or v-ATP-ase. These membrane-embedded proteins utilize the molecular energy contained in ATP to concentrate H+ ions in the intravesicular space. For each molecule of ATP used, one proton gets pumped into the vesicle.

Vesicular transporters pump molecules of neurotransmitter against their concentration gradients, which is an energetically difficult task. To have enough energy, the vesicular transporters use the high intravesicular concentration of H+ to move molecules of neurotransmitter across the vesicular membrane. When a proton moves from an area of high concentration to low concentration, energy is generated. The vesicular transporters use this energy to push neurotransmitter in. Because H+ ions move opposite of the neurotransmitter molecules, vesicular transporters are called antiporters. Transporters have slightly different stoichiometries, as it requires two protons to move a single molecule of dopamine, while the energy from a single proton is sufficient to transport GABA or glutamate.

Location of vesicles

Synaptic vesicles can be found in one of three places at the axon terminal.

1. Readily releasable pool (RRP). These vesicles are located close to the cell membrane at the axon terminal. In fact, many of them are already “docked,” meaning that their coat proteins are already interacting closely with the proteins on the inside of the cell membrane. When the depolarizing charge of an action potential reaches the terminal, these vesicles at the RRP are the first ones that fuse with the cell membrane and release their contents.

Figure 5.6 Synaptic vesicles in the axon terminal get filled by the action of two different vesicular transporter proteins. The v-ATP-ase uses energy to pump H+ into the vesicle against it’s concentration gradient (left). Then, a vesicular transporter such as vGluT use the movement of H+ down it’s concentration gradient to increase intravesicular concentration of neurotransmitter (right).

2. Recycling pool. These vesicles are the ones that have been depleted due to release. They are currently in the process of being refilled or reloaded with neurotransmitter. They are farther from the cell membrane, and the protein machinery is not primed for release, so it requires a more intense stimulation to release the contents of these vesicles.

3. Reserve pool. These vesicles are the farthest from the surface of the cell membrane, and most vesicles are held in this reserve pool. For these vesicles to be released, very intense stimulation is required. Reserve pool vesicles may not even be recruited for release under physiological conditions.

Release of vesicles

At a chemical synapse, the process of neurotransmitter release is very tightly regulated. If there were no mechanisms to control the release of chemicals at the synapse, nerve cells would deplete their entire stock of neurotransmitter. The signals that trigger muscle contraction at the NMJ would cause constant muscle tension. All sorts of brain signals would be active, and over-excitation would cause toxicity. Needless to say, regulated control of neurotransmitter release is a normal and essential part of nervous system function.

Regulation of release depends on several proteins that are important parts of the process. These proteins are often embedded within cell membranes of the vesicles or the neuronal membrane.

1. V-SNAREs are the proteins expressed on vesicles (v for vesicle). Synaptobrevin and synaptotagmin are two specific v-SNARE proteins that are involved during synaptic release.

2. T-SNAREs are proteins expressed on the cytoplasmic side of the axon terminal. The inside of the cytoplasm is the “target” for the vesicle (The t in t-SNARE). Syntaxin and synaptosomal nerve-associated protein 25 (SNAP-25), are t-SNAREs that function during vesicular fusion.

Figure 5.7 Axon terminals contain hundreds of vesicles roughly divided into three categories.

Figure 5.8 v-SNARE proteins interact with t-SNARE proteins to allow for vesicular fusion and release of neurotransmitter.

Clinical connection: Botulism

Botulism is a deadly condition that results from exposure to the spores produced by the bacteria Clostridium botulinum. Toxic spores can be found in the soil, contaminated foods, or water. The toxin itself is one of the most potent agents known - exposure to concentrations as low as 2ng/kg is lethal. The most common symptoms include muscle weakness or paralysis, especially the muscles of the face or the limbs. For about 5% of people who develop botulism, death results from paralysis due to respiratory failure.

Botulinum toxin is known to selectively cleave the proteins that comprise the SNARE complex. There are a few specific types of botulinum toxin with slightly different intracellular targets, but the result is the same on the molecular level: prevention of vesicular fusion eliminating neurotransmitter signals.

Despite being one of the deadliest toxins so far identified, millions of people pay to have a preparation of toxin called “Botox” injected into their face. For most, the injection of botox is a cosmetic procedure that can reduce the appearance of wrinkles by paralyzing the muscles. Botulinum toxin is also used medically for conditions resulting from excessive neurotransmitter release, such as muscle spasms, excessive sweating, or migraine.

Figure 5.9 Botulinum toxin selectively cleaves vesicular fusion proteins, preventing acetylcholine from being released at the NMJ.

Fusing of vesicles

The last step of neurotransmitter release is the fusing of the cell membrane. In order to release their chemical contents into the synapse, vesicles need to fuse with the cell membrane. As the vesicular membrane merges with the interior of the neuronal membrane, the contents of the vesicle become exposed to the extracellular space. Only then are the neurotransmitters capable of activating receptors.

One of the key proteins required for vesicular fusion is the vesicle-embedded V-SNARE synaptotagmin. This protein is capable of detecting elevated levels of Ca2+ in the axon terminal. As it turns out, an elevation of Ca2+ in the intracellular space is the “go ahead” signal that causes neurotransmitter release.

The concentration of intracellular calcium, generally in the range of 100 nM, is much lower than the concentration outside the cell. Embedded in the cell membrane of the axon terminals are transmembrane proteins called “voltagegated calcium channels” or VGCCs. As their name suggests, they function very similarly to the voltage-gated sodium channels described in previous chapters: they are large protein complexes that normally remain closed, but when the surrounding neuronal membrane becomes depolarized, they physically change conformation and open up, allowing ions to move across the cell membrane. These VGCCs selectively pass only Ca²⁺ ions. The electrochemical gradient causes these Ca²⁺ ions to enter into the cell.

As the change in membrane potential travels down the length of the axon (the action potential), it causes a depolarization at the terminal, triggering calcium entry via the VGCCs. Ca²⁺ at the terminal binds with synaptotagmin. The v-SNAREs and the t-SNAREs interact with one another in the presence of Ca²⁺, forming a molecular structure called a SNARE complex. The SNARE complex looks a lot like two twist ties that are wound tightly together. As they twist tighter together, it causes the vesicle membrane to approach the inside of the cell membrane, which results in vesicular fusion.

Vesicles are capable of fusing in at least two different ways.

1. Full fusion. A vesicle that undergoes full fusion experiences total exocytosis. The vesicular membrane becomes completely integrated with the cellular membrane, and all of the neurotransmitter spills into the synapse.

2. Kiss-and-run. This method of neurotransmitter release is incomplete fusion. The vesicle only partly connects with the interior surface of the cell membrane, and only a limited number of neurotransmitter molecules are able to enter the synapse via diffusion.

Figure 5.10 Synaptic vesicles either fuse completely (left) or partially in kiss-and-run (right).