membrane potential

International research collaboration reveals the mechanism of the sodium-potassium pump

It’s not visible to the naked eye and you can’t feel it, but up to 40 per cent of your body’s energy goes into supplying the microscopic sodium-potassium pump with the energy it needs. The pump is constantly doing its job in every cell of all animals and humans. It works much like a small battery which, among other things, maintains the sodium balance which is crucial to keep muscles and nerves working.

The sodium-potassium pump transports sodium out and potassium into the cell in a fixed cycle. During this process the structure of the pump changes. It is well-established that the pump has a sodium and a potassium form. But the structural differences between the two forms have remained a mystery, and researchers have been unable to explain how the pump distinguishes sodium from potassium.

Structure solves the mystery

Thanks to the international collaboration between Professor Chikashi Toyoshima’s group at the University of Tokyo and researchers from Aarhus University, the structure of the sodium-bound form of the protein has now been described. For the first time ever, the sodium ions can be studied at a resolution so high - 0.28 nanometres - that researchers can actually see the sodium ions and observe where they bind in the structure of the pump. In 2000, Professor Chikashi Toyoshima’s group described the structure of a calcium-pump for the first time, and in 2007 and 2009 research groups from Aarhus University and Toyoshima’s group described the potassium-bound form of the sodium-potassium pump.

“The new protein structure shows how the smaller sodium ions are bound and subsequently transported out of the cell, whereas the access of the slightly larger potassium ions is blocked. We now understand how the pump distinguishes between sodium and potassium at the molecular level. This is a great leap forward for research into ion pumps and may help us understand and treat serious neurological conditions associated with mutations of the sodium-potassium pump, including a form of Parkinsonism and alternating hemiplegia of childhood in which sodium binding is defective,” explains Bente Vilsen, a professor at Aarhus University who spearheaded the project’s activities in Aarhus with Associate Professor Flemming Cornelius.

Impressed Nobel Prize winner

The vital pump was discovered in 1957 by Professor Jens Christian Skou of Aarhus University, who received the Nobel Prize for his discovery in 1997. The new result is the culmination of five or six decades of research aimed at the mechanism behind this vital motor of the cells.

“Years ago, when the first electron microscopic images were taken in which the enzyme was but a millimetre-sized dot at 250,000 magnifications, I thought, how on earth will we ever be able to establish the structure of the enzyme. The pump transports potassium into and sodium out of the cells, so it must be capable of distinguishing between the two ions. But until now, it has been a mystery how this was possible,” says retired Professor Jens Christian Skou, who - even at 94 years of age - keeps up to date with new developments in the field of research which he initiated more than 50 years ago.

“Now, the researchers have described the structure that allows the enzyme to identify sodium and this may pave the way for a more detailed understanding of how the pump works. It is an impressive achievement and something I haven’t even dared dream of,” concludes Jens Christian Skou.

Membrane potential and Action Potential

Before you read about membrane potentials and action potentials you need to know the physics behind why they actually happen.  Even before you find out the physics behind it all, remember there is high [K+] INSIDE the cell and there is low [Na+] INSIDE the cell.  It’s opposite in the ECF.  This is why Na+/K+ Pump uses ATP.  It pumps 3 Na+ OUTSIDE the cell (where [Na+] is already high) and it forces 2 K+ into the cell (even though [K+] is already high inside the cell).

 

Basic Physics of Action Potentials

èA concentration difference across a selectively permeable membrane, can create a membrane potential.

 

If a membrane is permeable to only K+, than normally K+ would want to leak out of the cell.  Once it moves out, it will create a positive electro gradient outside the cell because the negatively charged proteins will stay inside the cell.  Diffusion potential will block further leakage of K+ ions out of the cell even though there is still higher concentration of K+ inside the cell.  Potential difference (in a regular mammalian nerve fiber) required is about -94mV.   I can use the same example using Na+, but Na+ would leak inside the cell, creates a positive electro chemical gradient inside the cell, until diffusion potential blocks the leakage, even though [Na+] is still higher outside the cell ect.  Potential difference (in a regular mammalian nerve fiber) is about +61 mV.

 

Nernst equation: can calculate the potential of any univalent ion at normal body temperature (98.6F/37C) è EMF (mV) = +/- 61 x log ( [inside]/ [outside]).  Assume that (1) temperature is normal levels (2)potential outside the cell = 0 (3) potential it measures is the inside of the cell.  + sign if ion is negative, - if the ion is positive.

 

When you want to measure different ions use Goldman’s equation.  The potential difference depends on (1) polarity of the ion (2) the permeability of the membrane (3) concentration of the membrane.

 

Resting Membrane Potential of Nerves

èThe total resting membrane potential is -90mV.

 

Remember the Potential difference of K+ (-94) and Na+ (+61) but since the membrane is 100 times more permeable to K+ than the potential difference  becomes (-86mV), which makes sense because it’s closer to K+’s (-94).  The Na/K Pump pumps 3Na out and 2K+ in with a net difference of -4.  The total resting membrane potential is -90mV.

 

Part of the factors that determine the level of resting potential is the active transport of Na+/K+ pump.  This pump is electrogenic meaning that it creates an electric gradient by pumping more positive ions out (3Na+ out/ 2K+in).  There’s also a leakage of K+ through the nerve membrane.  Potassium “leak” channels allow K+ to leak out even in a resting cell.  Na+ can also leak out but K+ is more permeable, so that doesn’t really happen much.  If you only account for the “leak” channels of K+ and Na+, then the resting membrane would be -86mV and K+ contributing more to the membrane potential than Na+.  But if you add the Na+/K+ pump which pumps 3Na+ out and only 2K+ in, than it will cause the membrane potential to be -90mV (add an extra -4 charge inside the cell).  In a resting cell the Na+/K+ pump is working so even in a resting cell, it still uses ATP.

 

èNerve Action Potential

Resting stage- the membrane is polarized -90mV

Depolarization stage- Is when Na+ suddenly becomes permeable and the membrane gains + charge because of it.  Sometimes certain cells overshoot the 0mV and go up to +35mV but in small fibers, and in many CNS neurons, the potential merely reaches 0.

Repolarization Stage- when the Na+ channels close and the K+ channels open more than normal.  The influx of K+ ions lets the membrane get back to original resting state.

·      Voltage-gated Sodium Channel- this channel has 2 gates.  One on the outside of the cell called the activation gate and one on the inside, the inactivation gate. At resting state the activation gates are closed.  When the resting state becomes about -70 to -50mV (threshold), there is a conformation change that will open the gate fully, the activation gate opens.  This is called the activated state and it increases Na+ permeability to 500-5000 fold.  The inactivation of the channel will close when the membrane potential reaches about +35, here the activation gate stays open while the inactivation gate closes.  The inactivation process means the inactivation gates will not reopen until the membrane potential becomes resting levels.

·      Voltage-gated Potassium Channel- when the membrane potential goes from negative 90mV close to 0, the K+ channels undergoes a confirmation change that will open the channel.  The decrease in Na+ entry and the simultaneous increase in K+ exit form the cell combine to speed repolarization.

 

èRoles of other Ions During Action Potential

Negatively charged ions (anions)(eg. Sulfate compounds, organic phosphate compounds, ect.) inside the nerve axon is one of the causes that makes the membrane potential negative.  Calcium ions are used in some cells in place of Na+.  Same thing happens to Ca++ like with Na+.  The Ca++ will pump Ca++ out the cell.  There is about 1000-fold Ca++ outside the cell than inside, so when voltage-gated Ca channels open, there will be a flow of Ca++ inside the cell.  These channels are also called slow channels because it takes 10-20 times longer for activation.  Ca++ are numerous in cardiac muscles and smooth muscles.  There is an increase in Na+ channels when there is a deficiency in Ca++.  When there’s a deficit of Ca++, the Na+ channels will become activated (open) by a small increase of the membrane potential from its normal, very negative level.  This causes the fiber to become highly excitable and cause spontaneous discharge.  This spontaneous discharge is called muscle “tetany” . This is sometimes lethal because it causes contraction of respiratory muscles.

 

Positive-feedback Cycle Opens the Sodium Channels.  When the potential from -90mV gets close to 0mV, then Na+ channels will start to open.  This will cause even more voltage-gated Na+ channels to open.  This is a positive feedback cycle that continues until all the voltage-gated Na+ channels are open.  A potential will occur when Na+ ions entering the fiber becomes greater than the K+ions leaving the fiber.  This doesn’t occur until about -65mV so this is called the threshold. 

 

èPropagation of the Action Potential

When a nerve fiber gets excited, Na+ will rush into a part of the cell.  This makes part of the cell reach -65mV threshold and cause an action potential, and then because of the positive charge in this area of the cell, it will open other Na+ channels around the area.  This is why an action potential travels along a nerve fiber.  It is called a nerve or muscle impulse.

 

The direction of propagation can go either way.  There all-or-nothing response means that an action potential will be administered only if all the conditions are met.  The ratio between action potential : threshold for excitation must be greater than 1. This is called the safety factor.

 

èRe-establishing Na+ and K+ ionic gradients after action potentials are completed—importance of Energy metabolism.

Re-establishing ionic gradients is due to the Na+/K+ pumps.  This “recharging” of the nerve fiber uses ATP.  The activity of the pump increases when there’s a lot of Na+ inside the cell.  If the [Na+] rises it can increase the pump’s activity eightfold.

 

èPlateau in Some Action Potentials

Sometimes the excited membrane doesn’t repolarize immediately after depolarization.  It sometimes remains on a plateau near the peak of the potential.  This type of action potential occurs in the heart muscle fibers.  The cause is a combination of (1) Na+ channels, fast channels (2) Ca++ channels, slow channels.  Opening of fast channels causes the spike portion of the action potential, and the prolonged opening of the slow Ca++/Na+ channels allows Ca++ to enter the fiber, which is largely responsible for the plateau portion of the action potential as well.  (3) The K+ channels are slower than usual to open. 

 

èRythmicity of Some Excitable Tissues – Repetitive Discharge

(1) heart (2) peristalsis of intestines (3) neuronal events of control , like breathing. Other tissues can discharge repetitively if the threshold for stimulation is reduced low enough.  Large muscle fibers can discharge repetitively when they are placed in a solution that contains the drug veratrine or when [Ca++] falls below critical value, both increases Na+ permeability of the membrane.

 

For spontaneous rhythmicity to occur, the membrane must be permeable enough to Na+ ions, or to Ca++ and Na+ ions (through the slow gates).  For the heart -60 to -70mV (1) Na+ and Ca++ flow inward (2) this increases membrane voltage in the positive direction which increases permeability (3) even more ions flow inward (4) permeability increases more until AP is generated.  Then the membrane repolarizes.  The AP doesn’t happen immediately after because of repolarization.  In this state, self-re-excitation cannot occur. 

 

Myelinated fibers causes salutatory conduction.  This increases the speed at which an action potential occurs and it also conserves energy.  Absolute refractory period is when an AP cannot be elicited even with a strong stimulus.