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17.3: Ligand and Voltage Gated Channels in Neurotransmission - Biology

17.3: Ligand and Voltage Gated Channels in Neurotransmission - Biology


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A. Measuring Ion Flow and Membrane Potential

When neurotransmitters bind to their receptors, ion channels in responding neuron or muscle cells open. The resulting influx of Na+ ions disrupts the resting potential of the target cell. The effect is only transient if the membrane potential remains negative. However, if enough Na+ ions enter the cell, the membrane becomes depolarized. If the cell experiences hyperpolarization, a localized reversal of normal membrane polarity (say from –70 mV to +65mV or more) will generate an action potential. This action potential will travel like a current along the neural or muscle cell membrane, eventually triggering a physiological response, e.g., the excitation of the next nerve cell in a neuronal pathway or contraction of the muscle cell. The patch-clamp device detects specific ion flow and any the resulting change in potential difference across the membrane. Principles of patch-clamp measurement are illustrated below.

In the example above, closing the switch on the power supply sends an electrical charge to the cell, opening up voltage-gated ion channel. In this case, a potassium sensor in the device detects the flow of K+ ions through the channel and out of the cell. At the same time, a volt meter registers the resulting change in membrane potential.

In addition to voltage-gated ion channels, the patch clamp device can measure ion flow through ligand-gated ion channels and mechanically-gated ion channels.

The former channels are receptor-ion gates that open when they bind an effector molecule. Mechanically-gated ion channels detect physical pressure or stress that result in a local membrane deformation, opening the channel.

Finally, cells maintain a high intracellular concentration of K+ ions, causing K+ ions to slowly leak from the cell, a phenomenon detectable by a patch-clamp. The presence of negative ions (Clions, organic ions) inside a cell limits the leakage. This creates the electronegative interior of a cell relative to outside the cell, i.e., the resting potential across its plasma membrane. The patch-clamp technique has been used to correlate the flow of ions and changes in membrane potential when a neuron fires, causing an action potential in a responding cell.

Such a correlation is described on the next page. In the illustration, follow the opening and closing of ion channels and the flow of ions. An action potential (in fact any shift from resting potential) results from facilitated diffusion of specific ions into or out of the cell through gated ion channels (green, above) that must open and close in sequence. The behavior of two different voltage-gated ion channels are illustrated in the graph. Electrical stimulation opens Na+ channels. Na+ ions rush into the cell, reducing the membrane potential from the resting state to zero, or even making the cytoplasm more positive than the extracellular fluid. If the reversal in polarity is high enough, a voltagegated K+ opens and potassium ions rush into the cell, restoring the resting potential of the cell.

A cell can continue to respond to stimuli with action potentials for as long as there is sufficient Na+ outside the cell and K+ inside the cell. While active transport of Na+ and K+ is not required to re-establish the resting potential, it will eventually be necessary to restore the balance of the two ions in the cell. If a nerve or muscle cell fires several times (or even if it just leaks ions), the [K+] inside the cell and the [Na+] outside the cell would drop to a point where the cell cannot generate an action potential when stimulated. Ultimately, it is the role of ATP-dependent Na+/K+ pumps to restore the appropriate Na+:K + balance across the responding cell membrane. As we have seen, each cycle of pumping exchanges 3 Na+ ions from the intracellular space for 2 K+ ions from the extracellular space. The pump has two effects:

  • It restores Na+ concentrations in the extracellular space relative to the cytoplasm.
  • It restores K+ concentrations in the cytoplasm relative to the extracellular space.

Together with the higher negative ion concentrations in the cytosol, the unequal exchange of Na+ for K+ ions maintains the resting potential of the cell over the long term and ensures that nerve and muscle cells remain excitable. Next, we will take a closer look at the role of both ligand-gated and voltage-gated ion channels in neurotransmission.

B. Ion Channels in Neurotransmission

Action potentials result in an orderly, sequential opening and closing of voltage- and ligand-gated channels along the neuronal axon. In the link below, you can see the sequential cycling of voltage-gated channels that propagates a localized action potential (membrane depolarization) along an axon towards a synapse.

When a propagated depolarization reaches a synapse, gated ion channels either open or close in the neuron and the responding cell. The cooperation of voltage- and ligand-gated channels at a neuromuscular junction is illustrated below.

As you can see from the illustration, after a neuron fires, an electrical impulse (a moving region of hyperpolarization) travels down the axon to the nerve ending. At the nerve ending, the traveling charge difference (electrical potential) across the cell membrane stimulates a Ca++ -specific voltage-gated channel to open. Ca++ ions then flow into the cell because they are at higher concentrations in the synaptic cleft than in the cytoplasm.

The Ca2+ ions in the cell cause synaptic vesicles to fuse with the membrane at the nerve ending, releasing neurotransmitters into the synaptic cleft. Then, the neurotransmitters bind to a receptor on the responding cell plasma membrane. This receptor is a ligand-gated channel (also called a chemically-gated channel). Upon binding of the neurotransmitter ligand, the channel opens. The rapid diffusion of Na+ ions into the cell creates an action potential that leads to the cellular response, in this case, muscle contraction. We have already seen that K+ channels participate in restoring membrane potential after an action potential, and the role of the sodium/potassium pump in restoring the cellular Na+/K+ balance.


Phases of action potential & role of gated ion channels

1. All cells have a membrane potential however, only certain kinds of cells, including neurons and muscle cells, have the ability to generate changes in their membrane potentials. Collectively these cells are called excitable cells. The membrane potential of an excitable cell in a resting (unexcited) state is called the resting potential, and a change in the resting potential may result in an active electrical impulse.

2 Neurons have special ion channels, called the gated ion channels, that

ahoy the cell to change its membrane potential in response to stimuli the cell receives. If the stimulus opens a potassium channel, an increase in efflux of potassium will occur, and the membrane potential will become more negative. Such an increase in the electrical gradient across the membrane is called a hyperpolarization. If the channel opened by the stimulus is a sodium channel, an increased influx of sodium will occur, and the membrane potential will become .less negative. Such a reduction in the electrical gradient is called a depolarization. Voltage changes produced by stimulation of this type are called graded potentials because the magnitude of change (either hyperpolarization or depolarization) depends on the strength of the stimulus: A larger stimuls will open more channels and will produce a larger change in permeability.

  1. In an excitable cell, such as a neuron, the response to a depolarizing
    stimulus is graded with stimulus intensity only up to, a particular level of depolarization, called the threshold potential. If a depolarization reaches the threshold, a different type of response, called an action potential, will be triggered.
  2. The action potential is the nerve impulse. It is a nongraded all-or-none event, meaning that the magnitude of the action potential is independent of the strength of the depolarizing stimulus that produced it, provided the depolarization is sufficiently large to reach threshold. Once an action potential is triggered, the membrane potential goes through a stereotypical sequence of changes.
  3. During the depolarizing phase, the membrane polarity briefly reverses, with the interior of the cell becoming positive with respect to the outside. This is followed rapidly by a steep repolarizing phase, during which the membrane potential returns to its resting level. Fig. 2.5.
  4. There may also be a phase, called the undershoot, during which the membrane potential is more negative than the normal resting potential. The whole event is typically over within a few milliseconds.

Role of gated ion channelgein the action potential:

The action potential arises because the plasma membranes of excitable cells have special voltage-gated channels. These ion channels have gates that open and close in response to changes in membrane potential. Fig. 2.4, 2.5

Two types of voltage-gated channels contribute to the action potential: potassium channels and sodium channels.

Each potassium channel has , a single gate that is voltage-sensitive it is closed when resting and opens slowly in response to depolarization.

By contrast, each sodium channel has two voltage-sensitive gates

(i) an ‘activation gate, that is closed when resting and responds to depolarization by opening rapidly, and

(ii) an inactivation gate, that is open when resting and responds to depolarization by closing slowly.

In the membrane’s resting state, the inactivation gate is open but the activation gate is closed, so the channel does not allow Na + to enter the neuron. Upon

depolarization the activation gate opens quickly, causing an influx of Na, which depolarizes the membrane further, opening more voltage-gated sodium channels and causing still more depolarization. This inherently explosive process. example of positive feed back, continues until all the sodium channels at the stimulated site of the membrane are open.

Two factors underlie the rapid repolarizing phase of the action potential as membrane potential is returned to rest. First, the sodium channel inactivation gate, which is slow to respond to changes in voltage, has time to respond to depolarization by closing, returning sodium permeability to its low resting level. Second, potassium channels whose voltage-sensitive gates respond relatively slowly to depolarization, have had time to open. As a result, during repolarization, K + flows rapidly out of the cell, helping restore the internal negativity of the resting neuron. The potassium channel gates are also the main cause of the undershoot, or hyperpolarization, which follows the repolarizing phase. Instead of returning immediately to their resting position, these relatively slow-moving gates remain open during the undershoot, allowing potassium to keep flowing out of the neuron. The continued potassium outflow makes the membrane potential more negative. During the undershoot, both the activation gate and the inactivation gate of the sodium channel are closed. If a second depolarizing stimulus arrives during this period, it will be unable to trigger an action potential because the inacthiation gates have not had time to reopen after the preceding action potential. This period when the neuron is insensitive to depolarization is called the refractory period, and it sets the limit on the maximum rates at which action potentials can be generated. Fig. 2.6


Ligand- and voltage-gated Ca 2+ channels differentially regulate the mode of vesicular neuropeptide release in mammalian sensory neurons

Neuropeptides released from dorsal root ganglion (DRG) neurons play essential roles in the neurotransmission of sensory inputs, including those underlying nociception and pathological pain. Neuropeptides are released from intracellular vesicles through two modes: a partial release mode called "kiss-and-run" (KAR) and a full release mode called "full fusion-like" (FFL). Using total internal reflection fluorescence (TIRF) microscopy, we traced the release of pH-sensitive green fluorescent protein-tagged neuropeptide Y (pHluorin-NPY) from individual dense-core vesicles in the soma and axon of single DRG neurons after Ca 2+ influx through either voltage-gated Ca 2+ channels (VGCCs) or ligand-gated transient receptor potential vanilloid 1 (TRPV1) channels. We found that Ca 2+ influx through VGCCs stimulated FFL and a greater single release of neuropeptides. In contrast, Ca 2+ influx through TRPV1 channels stimulated KAR and a pulsed but prolonged release of neuropeptides that was partially mediated by Dynamin 1, which limits fusion pore expansion. Suppressing the Ca 2+ gradient to an extent similar to that seen after TRPV1 activation abolished the VGCC preference for FFL. The findings suggest that by generating a steeper Ca 2+ gradient, VGCCs promote a more robust fusion pore opening that facilitates FFL. Thus, KAR and FFL release modes are differentially regulated by the two principal types of Ca 2+ -permeable channels in DRG neurons.


17.3: Ligand and Voltage Gated Channels in Neurotransmission - Biology

Ligand-gated ion channels form a pore through the plasma membrane that opens when a signaling molecule binds, allowing ions to flow into or out of the cell voltage-gated ion channels open in response to a change in membrane potential.

Ligand-gated ion channels bind a ligand, such as a neurotransmitter, and open a channel through the membrane that allows specific ions to pass through. To form a channel, this type of cell-surface receptor has an extensive membrane-spanning region. To interact with the phospholipid fatty acid tails that form the center of the plasma membrane, many of the amino acids in the membrane-spanning region are hydrophobic. Conversely, the amino acids that line the inside of the channel are hydrophilic to allow for the passage of water or ions. When a ligand binds to the extracellular region of the channel, there is a conformational change in the protein’s structure that allows ions such as sodium, calcium, magnesium, and hydrogen to pass through. The ligand-binding region is known as an allosteric site. It is located away from the actual ion channel but induces a conformational change in the channel to cause it to open.

One commonplace to find gated ion channels is in electrically excitable cells like neurons, which need to react very quickly to a stimulus. When a gated ion channel opens, it rapidly lets ions move through the channel, either into or out of the cell depending on the concentration gradient of ions across the plasma membrane (AKA the membrane potential). This converts an extracellular ligand signal into an intracellular electrical signal and will cause a change in the electrical properties of the cell.

Practice Questions

Khan Academy

MCAT Official Prep (AAMC

Biology Question Pack, Vol 2. Passage 4 Question 24

• Gated ion channels bind a ligand and open a channel through the membrane that allows specific ions to pass through.

• One common location to find gated ion channels is in electrically excitable cells like neurons, where they allow for a rapid change in the membrane potential of the cell.

• While ligand-gated ion channels rely on ligand binding to open, voltage-gated ion channels open in response to a change in membrane potential.

Membrane potential: the difference in electric potential between the inside and outside of a cell, defined by the concentration gradient of positively and negatively charged ions across the plasma membrane.

Allosteric site: a ligand-binding site on a protein that is located away from the active site (in the case of an enzyme) or the ion channel (in the case of a gated ion channel).

Ligand-gated ion channels form a pore through the plasma membrane

Neurotransmitters: chemicals that enable neurotransmission

Plasma membrane: separates the interior of the cell from the outside environment made of phospholipids

Hydrophobic: avoids water

Hydrophilic: likes water

Extracellular: outside the cell

Intracellular: inside the cell

Voltagegated channels: a class of transmembrane proteins that form ion channels that are activated by changes in the electrical membrane potential


Mechanically-gated channel:

This type of channel is opened upon bodily alterations by a simple sensation of touch.

The channels are located in the stereocilia of the interior region of the ear, wherein the sound waves are capable of bending the stereocilia and open the ion channel that results in the establishment of a nerve impulse.

Thus a stimulus in the arrangement of vibration or any physical pressure or stretching.

The gate thus represents a portion of the channel protein that opens or covers the pores.

The discrete states of the gate are either of conducting or non-conducting portions.

A Gated channel transports protein that unlocks a gate thereby permitting a molecule or ion to pass across its membrane.

The mechanically gated channels that open upon physical distortion of a receptor.


MATERIALS AND METHODS

Animals, cell culture, transfection, and plasmids

The use and care of animals were approved and directed by the Institutional Animal Care and Use Committee of Peking University and the Association for Assessment and Accreditation of Laboratory Animal Care. TRPV1-KO mice were provided by Z. Zhu (Third Military Medical University, Chongqing, China). Dyn1-KO mice were gifted by P. De Camilli (Yale University, New Haven, CT). Sprague-Dawley rats (

7 days old) were euthanized by an intraperitoneal injection of 0.15 ml of 10% chloral hydrate, and hypothermic anesthesia was used for mice. The DRGs of all spinal segments were isolated in ice-cold L15 medium (Gibco) and enzymatically dissociated in trypsin (0.2 mg/ml) and collagenase type 1A (1 mg/ml) containing Dulbecco’s modified Eagle’s medium/F12 for 40 min at 37°C. Cells were then dissociated by trituration and transfected with 3 μg of an NPY-pHluorin–expressing plasmid using a Neon (100-μl system) electroporation system (Invitrogen, MPK10096) according to the manufacturer’s instructions. The transfected cells were plated on polyethyleneimine-coated coverslips and cultured for 18 to 28 hours in a humidified incubator (37°C, 5% CO2) in Neurobasal-A medium supplemented with 2% B27 and 0.5 mM GlutaMAX-I (all from Gibco). NPY-pHluorin plasmid was constructed from NPY-Venus (a gift from N. Gamper, University of Leeds) and synapto-pHluorin (a gift from G. Miesenböck, University of Oxford). All chemicals were from Sigma, unless otherwise indicated.

Immunofluorescence

DRG neurons transfected with NPY-pHluorin were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature and permeabilized with 0.3% Triton X-100 for 5 min. Cells were blocked with 2% bovine serum albumin (BSA) in PBS for 1 hour at room temperature and then incubated overnight with primary antibodies diluted in PBS with 2% BSA at 4°C. After washing out the primary antibodies with blocking buffer, cells were incubated with Alexa Fluor 594–conjugated goat anti-rabbit immunoglobulin G (H+L) (Invitrogen, A11037) diluted in PBS with 2% BSA for 1 hour at room temperature. The nuclei were then stained with DAPI (4′,6-diamidino-2-phenylindole) and mounted with DAKO. Antibodies against CGRP (Peninsula, IHC6006) and secretogranin II (Abcam, ab12241) were used. Images were captured on an LSM 710 inverted confocal microscope (Carl Zeiss). For colocalization analysis, all intracellular puncta within 1-μm optical sections were selected and analyzed using the JACoP plugin of McMaster Biophotonics Facility ImageJ software (National Institutes of Health).

TIRF imaging, stimulation, and analysis

TIRF imaging was performed on an inverted microscope with a 100× TIRF objective lens (numerical aperture, 1.45 Olympus IX-81). Images were captured by an Andor electron-multiplying charge-coupled device using Andor iQ software with an exposure time of 50 ms. The standard bath solution contained the following: 150 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 10 mM H-Hepes, and 10 mM d -glucose (pH 7.4). The temperature was kept at

35°C throughout all TIRF experiments using a laboratory-made heater. Exocytotic events were defined as abrupt fluorescence increases, immediately followed by a fluorescence decrease or diffusion of NPY-pHluorin puncta in the vicinity. For the analysis of single release events, each event was selected and marked with 1.92-μm-diameter (center) and 2.4-μm-diameter (annulus) circular areas. Fluorescence intensity was calculated and analyzed using ImageJ the intensity values during the 0.5-s baseline before the peak value were averaged and used as F0. In FFL (or spreading) events, a robust fluorescence increase occurred at both the center and the annular area of NPY-pHluorin puncta, representing the release and spread of NPY-pHluorin. KAR events showed a brief brightening of the puncta, but no or only a very limited fluorescence increase in the annular area, representing a transient opening and reclosure of a restricted fusion pore that limited the release of NPY. High K + [85 mM NaCl, 70 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 10 mM H-Hepes, and 10 mM d -glucose (pH 7.4)] and capsaicin (300 nM) were applied using a gravity-fed perfusion system. Electrical field stimulation (1 ms, 15 V) was applied through a pair of platinum wires using an electronic stimulator (Nihon Kohden, SEN-3201) the negative pole was placed in the vicinity of the cell under examination. For two-color TIRF imaging, the Ca 2+ channels were labeled with mCherry (Cav2.2-mCherry and TRPV1-mCherry), and fusion events were visualized by NPY-pHluorin.

Fluorescence and fractional Ca 2+ measurements

Intracellular calcium ([Ca 2+ ]i) was measured using a Ca 2+ imaging system (TILL Photonics). Fura-2 potassium salt (1.0 mM) was loaded into the cell via a patch pipette in the whole-cell configuration. The fluorescence was sampled at 2 or 10 Hz.

Fractional Ca 2+ current, Pf, is defined as the percentage of Ca 2+ current in the total current passing through a cation channel (Im in this case). According to the original definition (40), P f = ∫ I Ca d t ∫ I m d t = Δ Fd F max × ∫ I m d t (1) where Im is the total whole-cell current and ICa is the proposed fractional Im current carried by Ca 2+ . ΔFd is the change of Fd, which is the “modified Ca 2+ -sensitive fura-2 signal” before (Fdt0) and after (Fdt1) the voltage pulse or ligand-induced Ca 2+ influx. Fd = F340 − F380, ΔFd = Fdt1 − Fdt0, and Fmax is a constant, which was determined by measuring the Ca 2+ influx through VGCCs in the solutions specified above. Under physiological conditions, all ions contributing to the current through VGCCs are Ca 2+ , namely, Pf = 100%. From Eq. 1, Fmax = ΔFd/∫ICadt, where ICa = Im (which is the current through VGCCs). According to Eq. 1, after determining the Fmax by measuring the fura-2 signal that is evoked after activation of TRPV1, the Pf of each channel can be determined.

CGRP immunoassay

Basal and stimulated extracellular CGRP concentrations were evaluated in freshly isolated DRG neurons using an enzyme immunometric assay kit (Bachem), following the manufacturer’s instructions. Cells were washed three times with normal external solution and then incubated in the same solution for 30 s at room temperature, followed by another 30-s incubation in this solution containing 70 mM KCl or 300 nM capsaicin. The incubation solutions were collected for subsequent analysis of basal and stimulation-coupled CGRP levels. All samples were centrifuged at 13,000 rpm for 5 min, and the supernatants were processed for CGRP measurement. Samples were analyzed at 450 nm using a microplate reader (BioTek Synergy 4). CGRP concentrations (in picograms per milliliter) were extrapolated from a best-fit line calculated from serial dilutions of a CGRP standard. All data points were measured in triplicate.

Electrophysiological recordings

We used an EPC10/2 amplifier with Pulse software (HEKA Elektronik) to obtain whole-cell patch-clamp recordings as described previously (41, 42). Pipette resistance was controlled between 3 and 4 megohms when filled with an internal solution containing 153 mM CsCl, 1 mM MgCl2, 10 mM Hepes, and 4 mM Mg–adenosine 5′-triphosphate (pH 7.2). Normal external solution contained 150 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 10 mM Hepes, and 10 mM glucose (pH 7.4). Igor software (WaveMetrics) was used for all offline data analyses. All experiments were performed at room temperature, unless otherwise indicated.

Calcium imaging

Changes of the [Ca 2+ ]i in DRG neurons were measured as the Fluo-4/Fura-Red fluorescence ratio (43). Cells were loaded with 2.5 μM Fluo-4 AM and 5 μM Fura-Red AM (Invitrogen) dissolved in 0.2% dimethyl sulfoxide and 0.04% Pluronic F-127 in a standard bath solution at 37°C for 20 min. Cells were then washed and imaged on an inverted confocal microscope (Zeiss LSM 710). The fluorescent Ca 2+ indicators were excited using a 488-nm laser, and the light emitted from the Fluo-4 and Fura-Red was recorded on separate channels at 500 to 540 nm and 600 to 680 nm, respectively. Images (512 × 512 pixels) were acquired at 1 Hz under a 40× oil objective lens (Zeiss). The Ca 2+ level was determined from ROIs using the ratio of intensity traces recorded on the Fluo-4 and Fura-Red channels.

Statistics

All experiments were replicated at least three times. Data were analyzed offline using ImageJ and Igor software. Data are means ± SEM. Statistical comparisons were performed using two-tailed unpaired Student’s t test, Kolmogorov-Smirnov test, or Mann-Whitney U test, as indicated. All tests were conducted using SPSS 13.0 (Statistical Package for the Social Sciences). Significance threshold was set at P < 0.05.


Contents

Voltage-gated ion channels Edit

Voltage-gated ion channels open and close in response to the electrical potential across the cell membrane. Portions of the channel domain act as voltage sensors. As the membrane potential changes, this results in changes in electrostatic forces, moving these voltage-sensing domains. This changes the conformation of other elements of the channel to either the open or closed position. [8] When they move from the closed position to the open position, this is called "activation." Voltage-gated ion channels underlie many of the electrical behaviors of the cell, including action potentials, resting membrane potentials, and synaptic transmission. [9]

Voltage-gated ion channels are often specific to ions, including Na + , K + , Ca 2+ , and Cl − . Each of these ions plays an important role in the electrical behavior of the cell. [9] The gates also have unique properties with important physiological implications. For example, Na + channels open and close rapidly, while K + gates open and close much more slowly. The difference in speed between these channels underlies the depolarization and repolarization phases of the action potential. [10]

Na + Channels Edit

Voltage Gated Sodium (Na + ) channels are significant when it comes to propagating the action potentials in neurons and other excitable cells, mostly being used for the propagation of action potential in axons, muscle fibers and the neural somatodendritic compartment. [11] Sodium(Na + ) channels are some of the main ion channels responsible for action potentials. [9] Being complex, they are made of bigger α subunits that are then paired with two smaller β subunits. [11] They contain transmembrane segments known as S1-6. The charged S4 segments are the channels voltage sensors. When exposed to a certain minimum potential difference, the S4 segments move across the membrane. [12] This causes movement of the S4-S5 linker, which causes the S5-S6 linker to twist and opens the channel. [13]

K + Channels Edit

Potassium (K + ) channels play a large role in setting the resting membrane potential. [9] When the cell membrane depolarizes, the intracellular part of the channel becomes positively charged, which causes the channel's open configuration to become a more stable state than the closed configuration. There are a few models of potassium channel activation:

  • The sliding helix model posits that the potassium channel opens due to a screwing motion by its S4 helix.
  • The paddle model posits that the S3 and S4 helices of the channel form "paddles" that move through the depolarized membrane and pull the S5 helix away from the channel's opening.
  • The transport model posits that a focused electric field causes charged particles to move across the channel with only a small movement of the S4 helix.
  • The model of coordinated movement of helices posits that the S4 and S5 helices both rotate, and the S4-S5 linker causes the S6 helix to move, opening the channel.
  • The consensus model is an average of the above models that helps reconcile them with experimental data. [14]
Ca 2+ Channels Edit

Calcium (Ca 2+ ) channels regulate the release of neurotransmitters at synapses, control the shape of action potentials made by sodium channels, and in some neurons, generate action potentials. [9] Calcium channels consist of six transmembrane helices. S4 acts as the voltage sensor by rotating when exposed to certain membrane potentials, thereby opening the channel. [15]

Neurotransmitters are initially stored and synthesized in vesicles at the synapse of a neuron. When an action potential occurs in a cell, the electrical signal reaches the presynaptic terminal and the depolarization causes calcium channels to open, releasing calcium to travel down its electrochemical gradient. This influx of calcium subsequently is what causes the neurotransmitter vesicles to fuse with the presynaptic membrane. [16] The calcium ions initiate the interaction of obligatory cofactor proteins with SNARE proteins to form a SNARE complex. [16] These SNARE complexes mediate vesicle fusion by pulling the membranes together, leaking the neurotransmitters into the synaptic cleft. The neurotransmitter molecules can then signal the next cell via receptors on the post synaptic membrane. These receptors can either act as ion channels or GPCR (G-Protein Coupled Receptors). [17] In general the neurotransmitter can either cause an excitatory or inhibitory response, depending on what occurs at the receptor.

Chloride channels are another group of voltage gated ion channels, of which are less understood. They are involved with processes such as skeletal and cardiac smooth muscle, cell volume regulation, the cell cycle, and apoptosis. [18] One major family of chloride proteins are called CLC proteins- common channels and transporters for basic physiological processes in mammals. CLC channels act as slow gated channels hydrogen ions are exchanged for an influx of chloride ions, allowing the anions to travel via their electrochemical gradient. [19] The voltage dependent C1C-1 chloride channel is homologous dimer which falls under this family, and is seen predominantly in skeletal muscle fibers. [20] With this channel the correct depolarization and repolarization via chloride ions is essential for propagation of an action potential. [18]

Ligand-gated ion channels Edit

Ligand-gated ion channels are found on postsynaptic neurons. By default, they assume their closed conformation. When the presynaptic neuron releases neurotransmitters at the end of an action potential, they bind to ligand-gated ion channels. This causes the channels to assume their open conformation, allowing ions to flow through the channels down their concentration gradient. Ligand-gated ion channels are responsible for fast synaptic transmission in the nervous system and at the neuromuscular junction. [21] Each ligand-gated ion channel has a wide range of receptors with differing biophysical properties as well as patterns of expression in the nervous system. [22]

Inactivation is when the flow of ions is blocked by a mechanism other than the closing of the channel. [8] A channel in its open state may stop allowing ions to flow through, or a channel in its closed state may be preemptively inactivated to prevent the flow of ions. [23] Inactivation typically occurs when the cell membrane depolarize, and ends when the resting potential is restored. [8]

In sodium channels, inactivation appears to be the result of the actions of helices III-VI, with III and IV acting as a sort of hinged lid that block the channel. The exact mechanism is poorly understood, but seems to rely on a particle that has a high affinity for the exposed inside of the open channel. [24] Rapid inactivation allows the channel to halt the flow of sodium very shortly after assuming its open conformation. [25]

Ball and chain inactivation Edit

The ball and chain model, also known as N-type inactivation or hinged lid inactivation, is a gating mechanism for some voltage-gated ion channels. Voltage-gated ion channels are composed of 4 [ dubious – discuss ] α subunits, one or more of which will have a ball domain located on its cytoplasmic N-terminus. [26] The ball domain is electrostatically attracted to the inner channel domain. When the ion channel is activated, the inner channel domain is exposed, and within milliseconds the chain will fold and the ball will enter the channel, occluding ion permeation. [27] The channel returns to its closed state, blocking the channel domain, and the ball leaves the pore. [28]

Deactivation is the return of an ion channel to its closed conformation. For voltage-gated channels this occurs when the voltage differential that originally caused the channel to open returns to its resting value. [29]

In voltage-gated sodium channels, deactivation is necessary to recover from inactivation. [24]

In voltage gated potassium channels, the reverse is true, and deactivation slows the channel's recovery from activation. [30] The closed conformation is assumed by default, and involves the partial straightening of helix VI by the IV-V linker. The mechanisms that cause opening and closing are not fully understand. The closed conformation appears to be a higher energy conformation than the open conformation, which may also help explain how the ion channel activates. [31]

Gating charge can be calculated by solving Poisson's equation. Recent studies have suggested a molecular dynamics simulation-based method to determine gating charge by measuring electrical capacitor properties of membrane-embedded proteins. [2] Activity of ion channels located in the plasma membrane can be measured by simply attaching a glass capillary electrode continuously with the membrane. [32] Other ion channels located in the membranes of mitochondria, lysosomes, and the Golgi apparatus can be measured by an emergent technique that involves the use of an artificial bilayer lipid membrane attached to a 16 electrode device that measures electrical activity. [32]


What are Ligand Gated Ion Channels?

Ligand gated ion channels are the second type of gated ion channels present in the cell membrane. The ligand is a small chemical molecule that interacts with the receptors of the channel proteins. They are a specific type of stimulating molecules. Once the ligand binds with the receptor, it will change the shape or the conformation of the channel protein.

Figure 02: Ligand Gated Ion Channels

The ligand gated channels will open so that the ions can easily enter or exit through these channels to or from the cell. Receptors can present in either the extracellular side or the intracellular side of the membrane. Acetylcholine receptors are one of the most studied ligand gated ion channels.


Conformational Mechanisms of Signaling Bias of Ion Channels

Ligand-gated Ion Channel Superfamily

Ligand-gated ion channels bind neurotransmitters and open in response to ligand binding. These channels control synaptic transmission between two neurons or between a neuron and a muscle. One subfamily encompasses the Cys-loop channels, so named because of a large extracellular domain containing Cys loops. 9,10 Members of this family include the channels that bind acetylcholine (the nicotinic acetylcholine receptor), GABA (the GABAA receptor), the 5HT3 receptor, and glycine receptors. Five subunits assemble to form the functional pentameric channel. Most are composed of two alpha subunits and three other subunits (beta, gamma, or delta) but some consist of five alpha subunits. Each subunit contains a large amino-terminal extracellular domain with a characteristic disulfide bond formed by a pair of cysteine residues (the Cys loop) and a transmembrane region formed by four helical segments (M1–M4). The channel pore is formed primarily by one of the helical segments (M4) from two of the subunits. The orthosteric ligand binding site is formed by the extracellular domain at the interface between subunits.

Another class of ligand-gated channel encompasses the tetrameric receptors for glutamate. 11 Glutamate is the major neurotransmitter in the mammalian brain and is largely responsible for excitatory synaptic transmission. The ionotropic glutamate receptor has three major structural domains: a large amino-terminal extracellular domain (ATD), an extracellular ligand binding domain (LBD), and a transmembrane region consisting of two helical segments per subunit. Binding of glutamate causes the “clamshell”- like LBD to close, resulting in opening of the pore. Several subtypes of ionotropic glutamate receptors exist, and can be broadly segregated based on their pharmacological sensitivity to glutamate-like molecules such as NMDA, kainate, and AMPA. As is typical for ion channel pharmacology, the distinct profile of these synthetic agonists in neuronal preparations foreshadowed the molecular identification of the three subfamilies of ionotropic glutamate receptor.

Yet another class of ligand-gated channel is characterized by a trimeric subunit arrangement. Each subunit has a large extracellular loop and two transmembrane segments. Members of this family include the acid-sensing ion channels (ASICs) that are gated by protons. 12,13 P2X channels, which are gated by extracellular ATP, are another example. 14,15


Sodium channels are primarily voltage-gated - these are the channels responsible for action potentials.

Many other receptors are ligand-gated, and these are typically the signal that causes the initial voltage change that opens the voltage-gated sodium channels however, these channels are less selective cation channels and are permeable to ions like potassium as well as sodium. Still, their permeability to sodium is quite important and so they may also be thought of as sodium channels sometimes.

These include neurotransmitter gated channels like nicotinic acetylcholine receptors and AMPA (glutamate) receptors, and transient receptor potential channels like the TRPV1 receptor that is sensitive to painful heat and the chemical capsaicin which makes chili peppers "hot."


Watch the video: Ion channels voltage gated, ligand gated, stress activated ion channel (May 2022).