Electrophysiology –A Simple VersionIt’s All about the Gates (Ion Channels)

Mike Clark, M.D.

Resting Membrane Potential

Every living cell has a charge on its membrane, termed the Resting Membrane Potential (RMP).

The RMP is due to cell membranes having passive leak channels (gates) in the membrane.

A passive leak channel is a transmembrane integral protein that is generally always open, allowing passive transport movement of certain ions into and out of the cell.

Which ions are of main interest in this discussion? Na+, K+, Cl-, and the large molecular Anions (A-)

Let’s say that the RMP sits in static positions (no lateral movement) on the cell membrane.

Certain cells (nerve and muscle) can laterally move a charge along a membrane; this is termed conductivity.

This conductivity is due to nerve and muscle cells being able to create Action Potentials.

Why can nerve and muscle cells create action potentials? They have Voltage Dependent Gates.

How do voltage dependent gates (channels) differ from passive leak channels? Voltage dependent gates are generally always closed and only open when the proper voltage is applied to the membrane.

The voltage needed to open a voltage dependent gate is termed Threshold Voltage.

Voltage Dependent Gates, like passive leak channels, are transmembrane integral proteins that allow the same ions to move in and out of the cell.

Are there other gates (channels) involved in electrophysiology? Yes, there are Ligand Gated Channels and some channels open and close due to mechanical factors, like pressure, some open and close in response to light (eyes) and there are other actions that open or close these cell channels in nerve and muscle.

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What is a Ligand? It is a generic term for any substance that can attach to a receptor.

Some ligands are chemicals known as neurotransmitter substances, some are hormones and there are also other chemicals.

Just as with Voltage Dependent Gates, the ligand gated channels are generally closed and open only when the proper chemical binds to the receptor.

Just as with all the mentioned gates, the ligand gated channels are transmembrane integral proteins that allow certain ions to flow through, either in or out.

What kinds of membrane potentials can ligand gated channels initiate? In nerve, they can generate Excitatory Postsynaptic Potentials (EPSP) or Inhibitory Postsynaptic Potentials (IPSP). In muscle they can generate Motor Endplate Potentials (MEPP).

The Resting Membrane Potential

What do cells need to have resting membrane potentials? (1) Cell membrane with transmembrane integral proteins acting as ion channels (2) These channels should remain open – leaky to these ions (3) The membrane should act as a resistor (physics) and capacitor (physics). (4) A constantly maintained unequal distribution of certain ions, where particular ions are not in equilibrium (Na+, K+, Cl-, and the large molecular Anions (A-)) from inside the cell to outside (5) The large molecular anions A- , must remain trapped inside the cell and not be allowed to leak out (6) The presence of an ATP dependent sodium/potassium (Na+/ K+) active transport pumps all along the membrane (thousands of them along the membrane).

Let’s examine some of the above factors. What is meant by a resistor? The membrane must resist the easy (not total) movement of the ions. The ions can leak through, but not at a rate they would like to. Why? The membrane is offering some resistance to them. A resistor in charge (electrical) physics is something that holds back a charge. Resistance can be measured in Ohms.

A capacitor holds a charge to a surface or surfaces. In this case the cell membrane holds a charge close to its two surfaces, where the net charge on the outside surface of the membrane is positive and the net charge on the inside of the membrane is negative.

Is the total net charge inside the cell negative and not just that on its surface? No, if we add up all the charges inside the cell they equate to a zero charge (Bulk Electroneutrality). The net negative inside charge is only on the surface, due to a dragging effect of charge.

Is the total net charge outside the cell positive and not just that on its surface? No, if we add up all the charges outside the cell, they equate to a zero charge (Bulk Electroneutrality). The net positive outside charge is only on the surface, due to a dragging effect of charge.

What are the normal distributions of ions involved? The discussion involves concentration differences. Point to note, when a chemical, like an ion is bracketed [] it means the concentration of that ion. So if you see this, for example [Na+], it means the concentration of sodium.

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Sodium [Na+] is an ion in higher concentration outside the cell than inside. The sodium concentration outside a cell is approximately 145 mM (millimole) and the inside 12mM. Potassium [K+] is an ion in higher concentration inside the cell than outside. The inside potassium concentration is approximately 145 mM and the outside approximately 4 mM. Chloride [Cl-] is an ion in higher concentration outside the cell than inside. The large molecular ions [A-] are totally trapped inside the cell, so its concentration is very high inside the cell compared to the outside. Any large molecular anions outside a cell are there because of damage to a cell membrane or released by neighboring cells that have died.

As a result of these unequal ion distributions, the cell has two gradients (electrochemical gradient) involving its cell membrane; concentration (chemical) gradient and a charge (electrical) gradient. This gradient is as a gradual change of both concentration and charge, like if you open a bottle of perfume, the aroma is high near the bottle and gradually dissipates as you move further from the bottle.

The concentration (chemical) gradient is a diffusion gradient, it involves the differences in concentrations of ions from inside to outside the cell and vice versa. Remember atoms and molecules move from a high concentration to low concentration in diffusion. Thus, sodium Na+ wishes to move into the cell; potassium K+ wants to move out of the cell; chloride Cl- wishes to move into the cell, and the large molecular anions A- wish to move out of the cell.

The charge (electrical) gradient involves the aforementioned charges held tight to membrane, where the outside of the membrane close to the membrane has a net positive charge and the inside of the membrane close to the membrane and net negative charge. As you know unlike charges like to move towards one another, so the positive outside charge would like to gradually (gradient) move towards the negative inside charge and vice versa.

Can all the mentioned ions (Na+, K+, Cl-, and the large molecular Anions (A-)) move through the membrane with the same ease? No

Chloride moves through the membrane the easiest, but potassium can almost move as fast through the membrane as chloride. However, the membrane does not allow sodium to move fast through it, so it moves much slower than chloride and potassium. The large molecular anions are not allowed to move through cell membrane channels at all.

Bulk Electroneutrality – what is it? Your textbook does not mention this, but for me this is a major factor in an understanding of the resting membrane potential. If you add up all the charges (positive and negative) inside the cell, they in-total come to a net charge of zero. If you add up all the positive and negative charges outside a cell, they cumulatively come to zero.

If the charges on the inside and outside of the cell come to a net zero charge, how does the outside of the membrane close to the surface have a net positive charge and the inside of the cell close to the membrane have a net negative charge?

This is due to a process that I will term the “dragging effect.” To begin to explain this, we have to understand the full desires of the movements of the ions. Let’s start with sodium Na+; sodium is a cation (positive charged). It has a strong desire to enter a cell, because it has a higher concentration outside the cell and the inside of the cell close to membrane has a net negative charge. Positive sodium would be pulled towards the negativity of inside of the cell, close to the membrane. The combined concentration and charge factors make sodium have the strongest moving force of all the ions. But

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remember, the cell membrane is not as permeable to sodium as it is to potassium. So, even though the forces say go into the cell sodium, the membrane resists this movement to a certain extent.

Speaking of potassium, let’s look at it. Potassium has a moderate desire to move out of the cell membrane. It does have a higher concentration inside the cell than outside, but the inside of the cell close to the membrane has a net negative charge. This net negative charge will tend to hold potassium back from moving through the membrane due to its concentration gradient. Thus, the concentration gradient and charge gradient are having opposing actions on potassium movement. Bottom line, potassium will not exit the cell with the same power as sodium wants to enter it.

OK, let’s put together the dragging effect. Let’s start with the strong desire of sodium ions to move inside the cell. When the positive sodium ion attempts to move into the cell, it cannot do it without requesting a negative ion to accompany it. Why? If a positive ion, like sodium, was allowed to enter the cell alone, it would add an extra positive charge inside the cell and violate bulk electroneutrality. Thus, the sodium ion must ask a negatively charged ion to enter with it. Chloride is a negatively charged ion that also wants to enter the cell, because it has a higher concentration outside the cell than inside. So, sodium is now ready to go along with chloride. Both sodium and chloride approach the membrane, but chloride can go through the membrane faster (it has better permeability). Chloride can get through the cell membrane using its passive leak channel. However, it gets caught in the middle of the membrane with only part of it tipping into the interior of the cell close to the interior surface of the membrane. Why is this? Because if it went all the way through it would add an extra negative charge to the cell interior (violate bulk electroneutrality). It then can only come in so far as it drags the positively charged sodium ion behind it. Thus, the positively charged sodium ion is pressed on the outside surface of the cell membrane, thus giving a net positive charge close to the outside surface of the cell membrane. At the same time, the chloride ion is held close to the inside surface of the cell membrane, giving a negative charge close to the membrane. When both sodium and chloride finally pop through (they will eventually do that) then a second set of sodium and chloride ions do the same thing and this is happening at locations all over the cell membrane. So this this then gives a constant net positive charge close to the outside surface of the cell membrane all over the membrane and a net negative charge close to the inside of the membrane all along the surface of the membrane. As I said the net charges are only along the surface of the membrane; the total charges on the inside of the cell an outside of the cell come to a net charge of zero.

Since the ions can passively leak according to their gradients, if allowed enough time, each ion type would reach equilibrium and there would no longer be a resting membrane potential. So, what sustains the resting membrane potential?

First understand that if the resting membrane potentials on your cell membranes are loss, you would die. This resting membrane potential is very important for many, many cell functions. One function to be discussed in this document is the action potential of nerve and muscle. An action potential is a moving potential, whereas the resting membrane potential is a more static non-moving charge. But, you cannot move a charge (action potential) without having a charge to move (resting membrane potential). If there are no action potentials, then there is no brain activity, no heart activity; no lots of activities.

The two things that maintain the resting membrane potential are the ATP dependent sodium/potassium pump and the intracellularly trapped large molecular anions.

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Let’s start first with what the intracellularly trapped large molecular anions do to contribute to maintain the RMP. The large molecular anions (A- ) contribute many negative charges to the interior of the cell. In order to maintain bulk electroneutrality, many cations (positive charged ions) must be present to negate the many anion negative charges inside the cell. It all is based on the fact the large molecular anions are trapped, so they cannot possibly move; they cannot ever reach possible equilibrium like the other ions can, due to passive leakage. Thus, their charged presence makes other ions not be able to reach their equilibrium because they must counteract the many negative charges they give. As mentioned, potassium ions desire to exit the cell due to their diffusion gradients, but many of them cannot exit the cell, because they must remain behind to counteract the negative charges of the trapped anions. On the other hand, negatively charged chloride ions desire to enter the cell, and they can move easily through the cell membrane. But, they cannot enter the cell mainly because there are already trapped negatively charged ions inside the cell, the large molecular anions. If chloride cannot enter, then sodium ions have difficulty entering because they need to enter with chloride and stated earlier.

What does the sodium/pump do?

The active transport sodium/potassium pump involves active transport. Point to note: anytime in physiology, the term pump is used, it automatically means an active transport action, requiring energy to operate from the breakdown (catabolism) of ATP. The pump pushes out of the cell 3 sodium ions into a region of high sodium concentration, while at the same time it pumps 2 potassium ions into the cell, into a region already high in potassium. Thus, the pump pushes out leaked sodium ions and pumps in leaked out potassium ions. This assists in maintaining the resting membrane potential, by keeping these ions at disequilibrium.

How do you measure the magnitude of the resting membrane potential?

The resting membrane potential involves a charge. The movement of charge is measured in volts. In electricity talk, volt measures the force of a current. Amps measure the how much flow and ohms is a measurement of resistance.

The measurement of the resting membrane potential is a measurement of the potential energy of charge movement. First of all, the ions want to move and have the energy to do it, but are being held back by the resistance (resistor) of the cell membrane. Thus, the ions are exhibiting potential energy (stored energy). In order to measure the ion potential energy, they must be allowed to move and express kinetic energy (energy in motion). How do we allow them to move? We give them a bypass route across the membrane. How do we do that? We place an electrode (charge measurement tool) inside the cell. The electrode is a thin hollow glass tube that can be filled with a charge solution (electrolyte solution) that can conduct current. This tiny thin electrode is simply pushed through the membrane and the soft membrane will seal around it. We take another electrode and place it close to the outside of the cell. A conducting wire (copper, gold, or other material) is placed in both electrodes, immersed into each electrodes charged conducting solutions. The wire is one long wire, but halfway of the wire a voltmeter is placed. So the flow of charge would come from the inside of the cell into the electrode charge solution, then carried along the wire, hitting the volt meter, then through the volt meter and on to the electrode conducting solution on the outside the cell. The charge went through the wire and bypassed the cell membrane. The force of the movement was measured in volts. The force of movement is minute, not enough for an entire volt; it is in thousandths of a volt, so it is in terms of millivolts.

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By international agreement, the intracellular positioned electrode is the measuring electrode and the extracellular electrode acts as a ground wire. Since the inside of the cell close to the membrane has a net negative charge, the RMP value is a negative value.

Each cell type has a different RMP value. For example, the neuron (nerve cell) has a usual RMP value of -70 mv (millivolts) and the value of the muscle cell is usually a – 90 mv (millivolts).

The Neuron Action Potential

Action potentials form the basis of nerve and muscle conductivity. This conductivity allows charges to move along the membrane, similar to how electricity moves along a wire. Action potentials have some similarity to a resting membrane potential, but many dissimilarities. The similarity is that ions (Na+ and K+) travel through transmembrane integral proteins; they travel through gates (channels) down their concentration gradients by diffusion (passive transport). The dissimilarities are (1) the gates are always closed and only open when a proper voltage (threshold voltage) is applied to the cell membrane (2) Each ion have their own specific gate; there is Na+ gate and a K+ gate (3) the gates are slightly larger than the passive leak gates, so ions can move through faster.

Preliminary Information to know about a neuron action potential1. The cell membrane is considered to be polarized. The outside of the cell membrane is a positive pole and the inside a negative pole.2. The voltage dependent transmembrane channel for sodium has two portions: an inactivation part that is always open at resting membrane voltage and an inactivation gate that is always closed until threshold voltage is reached (shown below). When threshold voltage is reached the activation gate quickly opens and the inactivation gate slowly eventually closes. 3. The same voltage that opens the activation gate and closes the inactivation gate also causes the opening of a voltage dependent gate to potassium, but it is very slow to open and close. 4. The action potential is all-or-none. This means the voltage height of the action potential gets no higher despite the magnitude of the threshold voltage. If the threshold voltage is a positive 40 mv, then no action potential will be created if less than 40 mv is given to the membrane; however, no greater sized action potential will occur if more than 40mv is delivered to the membrane.5. The total duration of a neuron action potential is about one millisecond. 6. The action potential graph has time in milliseconds on the x-axis and voltage in millivolts on the Y-axis.

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Steps of an Action Potential in a nerve cell (neuron)1. The cell membrane initially is at its Resting Membrane Potential.2. A voltage stimulus is given to the membrane that allows the cell membrane to reach threshold voltage at one location along the membrane.3. The threshold voltage causes a quick opening of the sodium activation gate and causes the sodium inactivation gate to slowly close. Because both gates are briefly open, sodium ions rush into the cell making the inside of the cell at that one location on the membrane become more positive. This positive rush in of sodium makes the membrane move to a zero voltage. Because of that the membrane is said to be performing depolarization. 4. Around the time that the inactivation sodium gate finally closes, disallowing any further sodium ions to enter the cell, the potassium voltage dependent gate opens and potassium ions begin to flow out of the cell. Because the positive potassium ions flow out of the cell, the inside of the cell again becomes more negative, bringing the cell membrane back to be more negative; this action repolarizes the membrane, termed repolarization. 5. The potassium gate was slow to open and is slow to close, thus it lets more potassium out than it should which gives a hyperpolarization; the cell is now more polarized that it was initially. In the drawing in this document, it is termed undershoot.

Absolute and Relative Refractory Periods

The action potential just described occurred on one spot of the neuron membrane. That one spot needs to rest before another action potential can be created there. The absolute refractory period is a time that it is absolutely mandatory for that spot on the membrane to rest and not perform another action potential there. The relative refractory period is a time another action potential could occur on that one spot, but the voltage needs to be higher than the usual threshold voltage.

What causes the absolute refractory period? The absolute refractory period occurs when the sodium voltage dependent gates have not been reset from the currently occurring action potential. Since the sodium gates are responsible for the initiation of the action potentials, until they are reset (activation gate closed and inactivation gate opened), new action potentials cannot initiate.

What causes the relative refractory period? When the potassium gates are open allowing potassium to rush from the cell, making the interior more negative, any stimulus to reach threshold has to be stronger. Why? The interior of the cell is getting more negative and threshold voltage requires the interior to get more positive. So the out rushing potassium is counteracting the reaching of threshold.

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How Does Conductivity Occur?An action potential occurs on one spot of the neuron cell membrane, but what causes it to be moved (propagated) along the membrane, like electricity along an electric wire.

Voltage dependent gates along a neuron cell membrane are very close together in the axon cell membrane; the axon is a portion of the neuron. The voltage change due to an axon potential creates a charged field around the spot of the action potential. The charged field causes a voltage change in the adjacent segment of the membrane; this voltage change is enough to reach threshold voltage in that area, thus starting an action potential in the adjacent area. The voltage change of the new action potential in the adjacent area of the membrane, causes the adjacent cell membrane next to it to start another action potential. This action repeats itself all along the cell membrane, thus moving the action potentials along the membrane, like electricity moving along an electric wire.

What is the all-or-none factor in action potentials and what is its significance?

The action potential is all-or-none. This means the voltage height of the action potential gets no higher despite the magnitude of the threshold voltage. If the threshold voltage is a positive 40 mv, then no action potential will be created if less than 40 mv is given to the membrane; however, no greater sized action potential will occur if more than 40mv is delivered to the membrane.

In order to see its significance, let’s examine the axon of a neuron.

The axon is a long structure and the action potentials must move all the way along the axon to its end.

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Let’s think of an analogy. Imagine that you had an opened out metal coat hanger and the fuse off a fire cracker. Cut the firecracker fuse the same length as the straightened out opened metal coat hanger. We then get a candle and a cigarette lighter. We touch one end of the metal coat hanger to the tip of the wick of the candle and use the cigarette lighter to heat up the other end. If we put our finger on the end of the coat hanger where it is being heated by the cigarette lighter it is very hot, but as we move our finger further away towards the end touching the candle wick, it gets cooler. It gets so cool that there is not enough heat to cause the candle wick to reach the kindling temperature, thus the candle wick does not light. The heat spread along the coat hanger is a passive spread of heat that diminished as you moved further away from the heat source.

On the other hand, we now do the same thing with the fire cracker fuse. Because of the construct of the fire cracker fuse, the heat of the cigarette lighter will cause the fire cracker fuse to light-up (burn) to the same extent all along the fuse. This means the heat will generate all along the fuse to the same extent; the candle wick being touched by the firecracker fuse will definitely light up. This is an all or none event; if the cigarette lighter had not lit up the firecracker fuse, nothing would happen, but if it did light it up the candle wick would light-up. The heat spread along the firecracker fuse is an active spread of heat that stays in a constant intensity all along the fuse due to the design of the fuse.

Because the action potentials are all-or-none, it ensures the action potentials will go to the very end of the axon with perfect intensity and then at a synapse activate whatever the neuron is hooked to.

Skeletal Muscle Resting Membrane Potential and its Action Potential

The muscle cell has a resting membrane potential of around a negative 90 millivolts (-90 mv), compared to a neuron RMP of a negative 70 mv. The muscle cell action potential involves voltage dependent sodium and potassium gates, just like the neuron. The skeletal muscle cell sodium voltage dependent gate does not have an activation gate and inactivation gate; it is just one gate. The gate opens when threshold voltage is reached and then eventually closes. The potassium gate is close to the same mechanism as that in a neuron. The skeletal muscle cell action potential is a little longer in duration than that of the neuron.

The action potentials travel along the skeletal muscle cell membrane just like they do with neurons, except the action potentials travel a little slower.

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Ligand Gated Channels

A ligand is any substance that can bind to a receptor. One type of ligand we will discuss is a neurotransmitter substance. Ligand gated channels oftentimes work to produce graded potentials, rather than the all-or-none action potentials. A graded potential opens according to the magnitude of the signal; the stronger the signal the more and longer the gate opens. Gated channels in neurons generally produce EPSPs (Excitatory Post Synaptic Potentials) and IPSPs (Inhibitory Post Synaptic Potentials). In skeletal muscle, a MEPP (Motor End Plate Potential) is created.

There are many neurotransmitter substances; each of them causes the opening or closing of certain ligand gated channels. The one involving skeletal muscle is acetylcholine. In order to discuss neurotransmitters, we must discuss chemical synapses. A synapse is an anatomical structure where a neuron connects to another neuron or a muscle or a gland. The two types of synapses are a chemical synapse and an electrical synapse. The least common synapse is the electrical synapse which involves one nerve or muscle cell hooking together by gap junctions. Gap junctions involve electrolytes moving freely between one cell and another. The electrolytes activate the next cell.

Let’s look at the anatomy of a chemical synapse. Example of neuroneuronal synapse!

Note the presynaptic membrane, the cell membrane before the synaptic cleft. The synaptic cleft is the space between the membranes; the neurotransmitter substance must cross this cleft. The postsynaptic membrane is after the synaptic cleft. The synaptic vesicles contain neurotransmitter substances and are in the presynaptic cell. The postsynaptic membrane has receptors for the neurotransmitter substance and it has the ligand gated channels.

How does the chemical synapse work?

Action potentials travel down the presynaptic membrane moving across the synaptic end bulb (labelled presynaptic bouton in this drawing). The action potentials, as they move across the synaptic end bulb, cause the opening of calcium (Ca++) ion channels found along the synaptic end bulb’s presynaptic membrane. Calcium ions, which are in higher concentration in the extracellular fluid, enter the presynaptic membrane by diffusion. This causes synaptic vesicles, which contain neurotransmitter substance, to be pulled to the intracellular surface of the presynaptic membrane. The synaptic vesicles fuse with the interior surface of the presynaptic membrane and open releasing neurotransmitter substance into the synaptic cleft by exocytosis. The neurotransmitter substance is released into the fluid

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Presynaptic Membrane

Synaptic Cleft

Postsynaptic Membrane

of the synaptic cleft; some is washed away but some is drawn to the receptors on the postsynaptic membrane. Receptors have specificity and affinity. The specificity means they only attach to a specific ligand and the affinity means “drawing power”; they pull the ligand towards them. Once the neurotransmitter substances attach to the receptors, they open ligand dependent gates. What happens then, depends on the specific neurotransmitter substances and the specific receptors. EPSPs, IPSPs or MEPPs could be formed.

EPSP (Excitatory Postsynaptic Potential)

An EPSP is not an action potential. It is a graded local potential caused by the opening of ligand dependent gates to sodium (Na+) ions in neurons. The sodium ions rush in through the ligand gate making the postsynaptic membrane more positive. The amount of the gates opening is due to the amount of stimuli, thus it is a graded opening unlike the all-or-none opening of action potentials. Various substances, such as neurotransmitter substances, hormones, and others can cause its opening. The purpose of the EPSP is to bring the postsynaptic membrane either to threshold voltage or closer to threshold voltage in order to initiate action potentials in the postsynaptic neuron.

IPSP (Inhibitory Postsynaptic Potential)

An IPSP is a graded local potential caused by the opening of ligand dependent gates to potassium (K+) or chloride (Cl-) or both ions in neurons. The potassium ions rush out through the ligand gates making the postsynaptic neuron’s membrane more negative or chloride ions rush in through the ligand gate making the postsynaptic membrane more negative. In both cases the postsynaptic membrane is made more negative. The amount of the gates opening is due to the amount of stimuli, thus it is a graded opening, like the EPSP. Various substances, such as neurotransmitter substances, hormones, and others can cause

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its opening. The purpose of the IPSP is to bring the postsynaptic membrane further away from threshold voltage. Motor Endplate Potential

The motor endplate on a skeletal muscle cell is the area on the cell reserved for a synapse to the motor neuron (somatic motor neuron). It is at this site that the neuromuscular junction is formed. It is at this site that the neurotransmitter substance, acetylcholine is released.

The motor end plate potential is similar to the EPSP in neurons; it is not an action potential, but rather a graded potential that involves the opening of ligand gated channels. The acetylcholine causes the opening of channels to both sodium and potassium, but due to the stronger driving force of sodium, more of it comes in thus causing the membrane to move to threshold; this allows adjacent voltage dependent gates along the skeletal muscle cell membrane to open initiating action potentials, which propagate in both directions along the membrane. These action potentials then travel along the membrane and into the T-tubules causing the release of calcium ions from the terminal cisternae; this starts the mechanical event of contraction.

Muscle has two major events: excitation and contraction. The excitation is the charge event, which involves the MEPP and the action potentials. The contraction is a mechanical event. The events must be coupled. Contraction does not occur without excitation. So, the buzz word is EC coupling (excitation/contraction coupling). Certain conditions and drugs can uncouple the two events.

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