Lesson 6 (5) Ion Channels and Transporters

What do channels and transporters regulate in cells?

The intra- and extra-cellular environments.

Where are ion channels and transporters located?

They span the cell membrane.

In which types of cells are many ion channels and transporters found?

in neurons, and some are found in glial cells.

What is important to note regarding ions?

The differences in ionic gradients.

What mechanisms bring about changes in Vm?

Both ion channels and active transport.

What does VmVm represent?

the membrane potential, which is a difference in electrical charge across the cell membrane (the charge inside is different than the charge outside).

What drives signaling within neurons, between neurons, and between neurons and other tissues?

Changes in ion distribution across the membrane drive signaling within neurons, between neurons, and between neurons and other tissues (for example, the neuromuscular junction).

By what mechanisms do changes in ion distribution (ion flux) occur?

Changes in ion distribution (ion flux) occur by way of both ion channels and active transporters (such as the Na-K pump)

What two essential components are required for signaling within neurons?

both active transporters and ion channels, which serve very different but essential purposes.

What is the function of active transporters (e.g., the Na^+K^+-pump) in neuronal signaling?

it creates and maintain ion gradients, establish the resting membrane potential (RMP), and reset the RMP after depolarizing events and action potentials.

What is the function of ion channels (e.g., voltage-gated Na^+-channel) in neuronal signaling?

They give rise to changes in membrane ion permeability that allow for ion movement, resulting in changes in membrane potential (Vm ). These changes are the basis of the action potential.

How do active transporters and ion channels complement each other in neuronal function?

transporters create the concentration gradients that help drive ion movements through open ion channels​

How does ion movement through a pump differ from movement through an open ion channel regarding conformational changes?

While an open ion channel allows unimpeded flow, a pump must undergo a series of conformational changes each time it moves an ion or ions.

Compare the speed of ion movement through a pump versus an open ion channel.

Ion movement through a pump is much slower (100 to 100,000 times slower) than through an open ion channel.

Does ion movement through an open ion channel require energy, and what about through a pump?

Ion movement through an open ion channel requires no energy. Ion movement through a pump requires energy, typically in the form of ATP, to transport ions against their electrical and chemical gradients.

How does ion movement through a pump differ from movement through an ion channel in terms of process type?

movement through the membrane by use of a pump is an active process, not passive as occurs through ion channels

What is rapid signaling in neurons dependent upon?

the rapid movement of ions through ion channels.

What drives the rapid movement of ions through channels?

both electrical and chemical concentration gradients.

Explain how diffusion and electrostatic forces drive ion movement across the neuronal cell membrane.

Diffusion is driven by a gradient, moving ions from a region of high concentration to a region of low concentration. Electrostatic forces drive ion movement because like charges repel and opposite charges attract.

When does ion flux occur across the neuronal cell membrane, and what drives it?

There is a constant movement of ions (ion flux) across the neuronal cell membrane, occurring both at rest and during changes in membrane potential. This ion flux is driven by both electrical and chemical concentration gradients.

What factors determine membrane permeability?

Membrane permeability is determined by the interaction of ions with water molecules, the lipid bilayer, and ion channels.

Why are membrane lipids described as hydrophobic?

Membrane lipids are hydrophobic because they do not mix well with water molecules.

Why are ions strongly attracted to water molecules, and what term describes them in this context?

Ions inside and outside the cell are strongly attracted to water molecules as such proteins are hydrophilic.

What are 'waters of hydration'?

Due to the partial charges (both + and -) on water molecules and the opposite charges on ions, ions in solution are typically hydrated

How do water molecules interact with cations and anions?

Cations are attracted to the oxygen atom of water, while anions are attracted to the hydrogen atoms of water, due to the partial charges on water molecules.

What is the energetic favorability of ions entering the lipid bilayer directly?

It is not energetically favorable for ions to leave the polar environment of water to enter the nonpolar environment of the lipid bilayer.

Why do smaller ions like Na^+ attract water more strongly than larger ions like K^+?

They have stronger electric fields due to the charge being more localized, causing them to attract water more strongly.

How does the hydration shell of Na^+ compare to K^+?

Na^+ ions have a larger water shell compared to K^+ ions, causing them to move more slowly.

How do potassium channels achieve selectivity?

They have a narrow pore that excludes the larger hydrated sodium ion.

How do sodium channels achieve selectivity?

They have a selectivity filter that stabilizes Na^+ by forming weak bonds. K^+ cannot be stabilized in this channel due to its larger size, and therefore cannot pass through.

What two factors determine ion permeability through selective channels?

charges (specific chemical interactions between the ion and the channel) and size (the diameter of the ion, which may or may not include water hydration).

What effect does adding the antibiotic gramicidin A have on artificial lipid membranes?

It results in small step-like changes in current.

What do the step-like changes in current across the membrane, caused by gramicidin A, reflect?

They reflect the all-or-none opening and closing of the single channel formed by the antibiotic.

How does the current (i) through a channel change if the electrical potential across the membrane (Vm) is varied?

The current (i) through the channel changes proportionally to the varied electrical potential (Vm).

When current through the channel (y-axis) is plotted against potential difference (x-axis) for a channel, what relationship is observed, and what law does it follow?

A linear relationship is observed between current flow and voltage, indicating that the channel behaves as an electrical resistor that follows Ohm's law (I = V/R).

What technique later confirmed the information about the linear relationship.

This information was later confirmed in biological membranes by the technique called patch-clamping​

What is patch-clamping?

it is a follow up or extension of voltage-clamping, allows one to observe single channel activity (i.e., it allows one to measure current flow through a single ion channel)

How is patch-clamping performed?

Briefly, a glass micropipette is placed over a part of membrane to isolate a single ion channel, with an electrode placed into the pipette to measure current flow through the channel.

What can be recorded using patch-clamping?

Small unitary current pulses that represent channel opening and closing can be recorded.

What relationship is observed between current and voltage in the ACh receptor using patch-clamping?

The relationship between current and voltage in the ACh receptor is linear.

Who developed voltage clamping, and what does it show?

Voltage clamping was developed by Hodgkin and Huxley, and it shows the entire/aggregate current flowing through thousands of channels.

Who developed patch clamping, and what does it enable?

Patch clamping was developed by Neher and Sakmann, and it enables one to measure current flowing through a single channel.

What was the significance of patch clamping in relation to Hodgkin and Huxley's proposals?

Patch clamping provided a means by which Hodgkin and Huxley’s proposals concerning ion channel characteristics could be tested directly.

In patch clamping, how is a seal created between the pipette and the membrane, and what is its purpose?

A slight suction is applied, creating a seal between the pipette and the membrane that prevents ions from flowing between the pipette and the membrane.

During patch clamping, what happens to the current when the channel is open, given the seal?

All current must now flow into the pipette when the channel is open.

What experimental control and characterization does patch clamping allow regarding membrane potential?

Patch clamping allows one to experimentally control the membrane potential to characterize the voltage dependence of membrane currents.

Is ion flux through ion channels active or passive, and does it require energy?

Ion flux through ion channels is passive, requiring no energy.

What determines the driving force, direction, and equilibrium of ion flux through ion channels?

The driving force, direction, and equilibrium are determined by diffusional and electrostatic forces.

What can change the net driving force for ion flux?

Changing either the electrical potential across the membrane or the concentration gradients can change the net driving force.

How does current flow vary with driving force in some channels, and what does this imply about their conductance?

In some channels, current flow varies linearly with driving force (where the channel acts like a simple resistor); here, the conductance is the same at all voltages.

What law does channel conductance follow when current flow is linear with driving force, and what is its formula?

Such channel conductance follows Ohm’s law, where I = V/R (note the linear relationship between I, current, and V, voltage).

How does current flow vary with driving force in rectifying channels, and what does this mean for their conductance?

In rectifying channels, current flow varies nonlinearly with driving force; here, conductance is variable over a given range of voltages.

How many conformational states do most ion channels have?

Most, if not all, channels have at least two conformational states: open and closed.

What controls the transition between the open and closed conformational states of an ion channel?

The transition is controlled by “gating”.

What physical changes in the protein structure are involved in channel “gating”?

Gating involves the protein channel undergoing a physical change in its structure—a twisting, tilting, or bending of an \alpha-helix, for example.

What is the typical purpose of these conformational changes in ion channels?

Such conformational changes typically serve to enhance conductance in the open state by making the channel pore wider and/or by making the channel pore more suitable with respect to charge—making it more polar via specific amino acids that line the pore.

What do ion channels generate?

Transient changes in electrical signals.

What are the 3 major mechanisms by which ion channels are controlled?

Ligand gating, voltage-gating, and phosphorylation gating.

Explain ligand gating for ion channels.

In ligand gating, the ligand acts as a chemical agonist that can bind to the extracellular or intracellular site. Neurotransmitters bind to an extracellular site, while Ca^{2+}, cyclic nucleotides, and GTP-binding proteins bind to an intracellular site.

What is the source of energy for opening channels in phosphorylation gating?

The energy for opening the channel comes from the transfer of the “high-energy” phosphate group.

Besides the major gating mechanisms, what other factors can regulate some channels?

Some channels are regulated by stretch orpressure changes (SEE Fig 5-6D)​

What happens to many voltage-gated channels after depolarization and opening?

Many voltage-gated channels inactivate (become refractory) after depolarization and opening.

How is inactivation in voltage-gated channels (such as for Na^+ and K^+) thought to occur?

Inactivation in voltage-gated channels is thought to result from conformational changes separate from that for activation.

When do many voltage-gated channels recover from the refractory state?

Many voltage-gated channels recover from the refractory state—returning to the resting state—only after the membrane potential is restored to its resting value.

How do some voltage-dependent Ca^{2+} channels inactivate after the internal Ca^{2+} concentration has increased?

The Ca^{2+} binds to calmodulin, a calcium-binding protein associated with intracellular signaling.

How do drugs and toxins act as exogenous factors on channel activity?

They can block (competitive binding) or enhance (noncompetitive binding) channel activity.

What is characteristic about some drugs and toxins binding to channels?

Some may bind to the same site as the endogenous ligand and can be either reversible or irreversible.

Give examples of exogenous ligands that bind to the nicotinic acetylcholine receptor (nAChR) and describe their binding characteristics.

Curare binds weakly and reversibly whereas \$\alpha\$-bungarotoxin binds strongly and irreversibly.

Some exogenous ligands bind in a noncompetitive fashion to influence channel gating and activity, what does that do?

Such binding typically doesn’t interfere with normal neurotransmitter binding, but will alter the response of the channel when its natural ligand is bound.

Provide an example of a drug that noncompetitively influences channel activity.

Binding of diazepam (Valium) to a regulatory site on chloride channels gated by GABA will prolong channel opening, in response to GABA binding.

How can ion channels be constructed?

Ion channels can be constructed as hetero-oligomers, homo-oligomers, or from a single polypeptide chain organized into repeating motifs that span the membrane.

What are the subunits of ion channels composed of?

The subunits of ion channels are complex proteins composed of amino acids.

What else do some chanels do

In addition, some channels contain auxiliary subunits that modulate channel gating

How many α-helix regions does each subunit of a nAChR have, and where are they located?

Each subunit of a nAChR has 4 α-helix regions (M1-M4) that span the membrane (this is 1 of 5 subunits composing the receptor)​.

What are the characteristics of each α-helix in a nAChR subunit concerning length and terminal locations?

Each α-helix is about 20 amino acids in length, with both the -NH_2 (amino) and -COOH (carboxyl) terminals located on the extracellular side.

What is a hydrophobicity plot used for, and how are regions denoted by convention?

A hydrophobicity plot is used to determine the degree of attraction to water of each region or segment. By convention, more hydrophobic regions are denoted “+” and more hydrophilic regions are denoted “-”.

What information is reflected in a hydrophobicity plot, and what is a hydropathic index?

The hydrophobicity plot reflects the hydrophilic or hydrophobic nature of the side chains of amino acids, with each amino acid given a hydropathic index shown on the y-axis of the plot.

What is the structure of ligand-gated channels that are receptors for ACh, GABA, glycine, and serotonin?

They all have 5 subunits, each subunit composed of 4 transmembrane \$\alpha\$-helixes. (the glutamate receptor belongs to a different family)

What is the composition of gap junctions, which connect the cytoplasm of two cells?

Gap junctions are composed of 12 identical subunits, each of which has 4 transmembrane domains or segments.

Describe the core motif of voltage-gated channels and the function of the P-region.

Voltage-gated channels have a core motif composed of 6 transmembrane segments (S1-S6) with the S5 and S6 segments connected by a P-region, which forms the selectivity filter of the channel.

What is characteristic of electrical synapses?

Electrical synapses involve direct cell-to-cell communication.

How do gap junctions compare to chemical synapses in number, and where are they found?

Gap junctions are fewer in number than chemical synapses, but are found in virtually all nervous systems.

What do gap junctions permit in terms of electric current flow?

They permit the direct, passive flow of electric current from one neuron to another.

How do gap junctions connect neuronal membranes?

They connect two neuronal membranes both structurally and functionally, connecting their two cytoplasms.

What type of synapses are gap junctions, and where do they occur?

They are intercellular electrical synapses that occur between a presynaptic membrane and a postsynaptic membrane

What structures compose gap junctions, and where are they present?

Gap junctions are composed of connexons, which are present in both pre- and postsynaptic membranes.

How is each connexon in a neuronal membrane formed?

Each connexon in each membrane is formed by six connexin subunits.

How many connexin subunits form a complete gap junction between two neuronal membranes?

A gap junction between two neuronal membranes is formed by 12 connexin subunits (six from each membrane, forming a connexon in each).

How does the size of a gap junction pore compare to that of a voltage-gated channel pore?

Gap junctions form a pore that is much larger than the pore of a voltage-gated channel.

How do substances move between the cytoplasm of pre- and postsynaptic neurons?

Substances simply diffuse between the cytoplasm of the pre- and postsynaptic neurons.

What types of substances can flow passively through gap junctions?

This flow includes ions as well as the passive movement of large molecules like ATP and second messengers like cAMP.

What are microscopic currents?

Currents that flow through single ion channels. (Patch clamping)?

What are macroscopic currents?

Currents that flow through a large number of ion channels.

How do microscopic and macroscopic currents differ in magnitude?

Microscopic currents are much smaller than macroscopic currents.

How are macroscopic currents typically measured?

Using voltage clamp techniques.

• Depolarizing pulses (A) applied to apatch of membrane containing asingle Na+ channel produces briefinward currents (B)​

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• Averaging such currents shows thatmost of these channels open withinthe first 1-2 milliseconds afterdepolarization (C)​

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• A macroscopic current (fromanother axon) shows the closecorrelation between the timecourses of microscopic andmacroscopic Na+ currents (D)​

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• The probability of Na+ channelopening depends on the membranepotential, increasing as themembrane is depolarized (E)​

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What is the effect of depolarizing pulses on a patch of membrane containing a single Na^+ channel?

They produce brief inward currents.

When averaged, how do single Na^+ channels typically respond to depolarization?

Averaging the current measurements shows that most of these channels open within the first 1-2 milliseconds after depolarization.

What does a macroscopic current demonstrate regarding Na^+ currents?

it shows a close correlation between the time courses of microscopic and macroscopic Na^+ currents.

What factor influences the probability of Na^+ channel opening?

it depends on the membrane potential, increasing as the membrane is depolarized.