“To Be or Not to Be … an Inhibitory Neurotransmitter by Frank Miskevich, Department of Biology, University of Michigan-Flint__________________________________________________________________________________________________________________________________________________________________

___

“Why so glum, Jessica?” George asked as he walked into the lounge.

“It’s my thesis experiments,” Jessica replied, throwing her pencil down in disgust. “They aren’t making any sense.”

George laughed. “Well at least you get to stay inside and look through a microscope! Larry was sitting outside for four hours in the rain counting grackles yesterday. What kind of cells are you looking at again?”

“Neurons. I finally got them to grow in a dish and can record from them, but my data are really weird.”

“You must need a small Microphone torecord them chatting away.”

“Not sound,” Jessica replied. “Yourecord electrical activity with asmall electrode stuck into the cell.Every time I stimulate them with neurotransmitter I get somespikes.” She flipped through a couple of open windows on herlaptop. “Like this one here. It’s called a trace.”

3 sec

40 mV

Clicker Question #1

“So how can neurons carry electrical signals?” George asks innocently.“I’ve heard of dendrites and axons and stuff, but it never made muchsense to me. Aren’t axons and dendrites just like wires that connectto each other using chemical signals?”

Jessica answers:A. they use Morse code--where do you think that came from?B. cells have tiny metal wires going throughout the cell.C. they use positive and negative ions moving through protein channels scattered over the whole length of the cell.D. they bring positive ions in through dendrites and negative ions in through axons.E. they bring negative ions in through dendrites and positive ions in through axons.

Click to review neurons and ions.

“Wait a minute,” George said. “That doesn’t make sense. I thoughtmembranes were supposed to keep ions and stuff like that in cells.”

“They do,” Jessica agreed, “but proteins can help molecules movethrough a membrane. Our cells have a lot of different proteins ontheir membranes, especially neurons. Some of us may even havemore neurons than others.”

Na+

Na+

Na+

Na+

Na+Na+

Na+

Na+

Na+

Ca2+K+

K+

K+

K+

K+

K+ K+

K+

Cl-

Cl-

Cl-

Cl-

Cl- Cl-Cl-

Cl-Cl-

Cl-

Cl-

Cl-

Ca2+

Ca2+

Ca2+

Clicker Question #2

“OK then, brainiac, why would ions want to move into a neuron if you dump neurotransmitter on it?” George demanded sarcastically.

A. Because ions bind hormones and hormones like to enter cells.

B. Because cells can engulf things like ions and bring them in.

C. Because positive ions always move into a negatively charged cell.

D. Because negative ions always move into a positively charged cell.

E. Because ions move through channels according to their electrochemical gradient.

“Electrochemical gradient? Sounds like chemistry to me,” Georgesaid. “Are you telling me it works justlike diffusion of a dye in water?”

Jessica smiled. “Exactly! Except it’s not just concentration thatmoves the ions but also electrical charge. Neurons are normally negatively charged on the inside. They spike when they become more positive.”

Click to review types of diffusion.

“If you say so. Then I guess you cause neurons to spike by dumpinga neurotransmitter onto the cells?”

“Yep. The one I’m studying is known as GABA,” Jessica replied. “Itusually makes neurons more negative and keeps them from firing.”

Click to review electrochemical gradient

Clicker Questions #3

George looks interested. “So what kind of protein lets negative ions in when you add a chemical neurotransmitter?”

A. Ligand gated channels

B. Voltage gated channels

C. Mechanosensitive channels

D. Uniport transporters

E. Co-transporters

Click to reviewprotein channels.

Click to reviewprotein transporters.

Clicker Question #4

George looked skeptical. “OK, I get that a channel works like a gateto let ions into or out of the cell depending upon conditions. Then what?What happens to the ions after they move into the cell?”

Jessica smiled serenely and replied:

A. other ions then help move them out and they go on from there.

B. ATP is used by various pumps to push ions back out of the cell.

C. ATP binds to the ions and carries them back out of the cell.

D. a different channel opens and lets the same ions move back out.

E. so few ions cross the membrane that concentrations do not change enough to matter.

Click to review active transporters

George nodded. “I get it. When you put, what was it, GABA, on yourcells, channels open and let ions in. So what kind of ion does GABAlet into a cell?”

“Chloride. When more chloride goes into a neuron, it makes cells more negative and therefore they shouldn’t spike as well.” Jessica sighed.

“Fair enough. So what’s your problem?”

Jessica looked deflated. “GABA is supposed to make cells morenegative, which keeps them from spiking. In some of my cultures, it’sdoing the exact opposite. At day 9 it seems to make them more positive and causes them to spike. All of my other neurotransmitters work the same way at both ages. Here’s the data. See for yourself.”

adding glutamate

adding GABA

adding acetylcholine

adding glutamate

adding GABA

adding acetylcholine

culture day 9

culture day 14

neurotransmitter additions

3 sec

40 mV

George whistled. “I see what you mean. Day 9 neurons are spikingwhen you add GABA. That’s not supposed to happen, is it?”

“No, it isn’t,” Jessica snarled. “I’ve done the experiment five times now, and every time I try it I see the same thing. Young neurons spike in response to GABA. I don’t get it.”

“Are the neurons making a different GABA channel?” George asked.“A different protein might respond differently.”

“I thought of that,” Jessica replied. “I checked my cells, and theexact same GABA channel is there at both ages. If the same proteinis there then it should react the same way.”

George asked, “Can other ions go through channels activated by GABA?”

“No, they’re specific,” Jessica answered. “I even looked for other GABA channels that might be made here. There aren’t any. I don’tknow what to do next.”

George thought for a minute. “It’s a tough one, all right. Are there any ther proteins that might be different?”

“Only 25,000 or so,” Jessica moaned. “I can’t go looking for a needlein a haystack. I need to graduate this May!”

“Well, I’m not really a scientist, so I can’t help you. But there must besomething different between them. Maybe the chloride doesn’t like the smell inside the young neurons?”

“Ions can’t smell, George,” Jessica responded.

“Well something must be stimulating those neurons. I still thinkthe chloride ions don’t like it in the cell for some reason. When in doubt,go back to the basics I always say. Anyway, I gotta run. Will I see youat the party next Friday?” George asked.

“Not unless I can figure this thing out before then,” Jessica grumbled.

Clicker Question #5

Which first principle will control the direction of a chemical reactionsuch as ion flow across a membrane?

A. EntropyB. EnthalpyC. Free energyD. Uncertainty principleE. Newton’s first law of motion

On Friday, Jessica walked up to George at the party. “Hi George.You know, you’re smarter than you look.”

“Of course I am. Ummm, exactly how am I smarter?”

“You told me to go back to first principles.” Jessica smiled. “Itworked! Have you ever heard of free energy?”

“As opposed to slave energy”" George asked, looking puzzled.

“Ha ha. Spoken like a true historian. No, free energy is what makesions move in a particular direction across a membrane.”

“OK,” George asked after a long pause, “so how does it do that?”

Click to reviewDG equations

“It all comes from this one equation.”

George stepped back. “Don’t you go pointing that equation at me.I left math a long time ago and don’t want to go back.”

Jessica smiled. “OK, I’ll just show you the details. I promise not tomake you do the calculations. In words, free energy is determinedby two things: the concentration of the ion on both sides of themembrane...”

“Right, diffusion,” George interrupted.

“Yes. Both concentration and the electrical properties of the cell andthe ion. Here, let me show you.” Jessica pulled out her mobile phoneand brought up a picture.

DG = RT*ln[Sc]in/[Sc]out + zFVp

excitationinhibition

-70 mV to-80 mV

-70 mV to-40 mV

Cl-

Cl-

Na+

K+

Ca2+

Jessica explained, “If GABA is working as an inhibitory transmitter,the neurons should become more negative. This is what they are doingin the older neurons. If GABA is working to excite neurons, thenchloride must be flowing out of the cell. Follow?”

“Barely, but go ahead,” George said.

“So I thought about free energy and the equation. One way to makechloride enter a cell is to lower the intracellular concentration ofchloride when it get older. There are only a few proteins that cando that, so I started looking.” Jessica smiled. “Have you ever heardof a protein called KCC2?”

George stared, waiting. “You don’t really expect an answer, do you?”

GABA

Cl-

young neurons

GABA

Cl-

KCC2

old neurons

“KCC2 is a symporter, and carries potassium and chloride ions outof the cell. I don’t see any effects based on potassium, but if chlorideis lower on the inside of the cell, GABA will open the same exactchannel on the cell surface and chloride will flow into the cell insteadof out of the cell. KCC2 is only found in older neurons. Here’s thesituation. For free energy, we only worry about chloride.”

-70 mV overall 10 mM Na+

120 mM K+

20 mM Cl-

extracellular150 mM Na+

2 mM K+

150 mM Cl-

KCC2-70 mV overall 12 mM Na+

115 mM K+

8 mM Cl-

young neurons old neurons

DG= RT*ln([Clin]/[Clout]) + zFVp

DG= 1.987*310* ln (0.020/0.15) + (-1)*23062*(-.07)

DG= -1241 + 1614

DG= +373 cal/mol, so the result is that chloride ions flow out of the cell rather than into it.

- in very young neurons, GABA is an excitatory neurotransmitter

- negative charges flowing out makes a cell more positive

GABA

Cl-

DG= RT*ln([Clin]/[Clout]) + zFVp

DG= 1.987*310* ln (0.008/0.15) + (-1)*23062*(-.07)

DG= -1806 + 1614

DG= -192 cal/mol, or now flows INTO the cell

- changing the intracellular chloride concentration converts GABA from an excitatory neurotransmitter to an inhibitory one

- neurons require very low intracellular chloride for neurotransmitters to be inhibitory

- neurotransmitters start out excitatory, and become inhibitory over time

GABA

Cl-

KCC2

George looked impressed. “So by lowering the chloride concentrationin older neurons, KCC2 turns GABA into an inhibitory transmitter.Is that useful for anything?”

Jessica smiled happily. “It seems to be important for making synapsesinitially. If target cells don’t become excited, they don’t know thatan axon is trying to make contact with them.”

George grinned at Jessica. “Hey, I’m all for making contact. Are youbusy later tonight?”

Jessica grinned back. “Sorry, George, I’m an old neuron. It takes alot more than that to get me excited!”

• Neurons in particular spend a lot of energy controlling their ions.

• The “electrical signals” neurons use to carry information are all based on the controlled flow of ions across the membrane at the right time.

• Balance of the various ions is critical for the neuron to function.

How can this neuron control it’s ion balance?

Which ions are most important?

Neurons in Action

Transport Across Membranes

• All cells regulate the materials, particularly ions, that travel across the cell membrane.

• Membrane transporters, channels, and pumps work together to maintain the ion concentrations inside the cell.

Na+

Na+

Na+

Na+

Na+Na+

Na+

Na+

Na+

K+

K+

K+

K+

K+

K+ K+

K+

Cl-

Cl-

Cl-

Cl-

Cl- Cl-Cl-

Cl-Cl-

Cl-

Cl-

Cl-

Ca2+Ca2+

Ca2+

Ca2+

Neurons and Neurotransmitters

• Neurons must have tight control over their ion balances.

• They use “electrical signals,” which are really changes in membrane potential, to carry information.

• Neurons carefully regulate their resting potential to around -70 mV using multiple different ion pumps and channels.

K+

K+

K+

K+

K+

K+

K+

K+

K+

K+

Na+

Na+

Na+

Na+

Na+

Na+

Na+Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Na+

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl- Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

• Excitatory neurotransmitters depolarize neurons (move more positive).

• Inhibitory neurotransmitters hyperpolarize neurons (move more negative).

• Neurotransmitters allow ions to flow through ligand gated ion channels known as neurotransmitter receptors.

• The direction of the ion flow determines whether the neurotransmitter is excitatory or inhibitory (note the importance of Cl- for inhibition!)

Neurons and Neurotransmitters

excitationinhibition

-70 mV to-80 mV

-70 mV to-40 mV

Cl-

Cl-

Na+

K+

Ca2+

return to the case

Membrane potential: relative net charge on opposite sides of a membrane.

Typical cell membrane potential, or resting potential, is ~ -60 mV insides of cells are more negative.

Electrochemical gradient: combined electrical and chemical free energies for a given ion.

+

+

+

+

+ +

+

+

-

-

-

-

--

--

- -

-

outside inside

+

+

-

-

return to the case

Why Molecules Move Across Membranes

Transport Across Membranes

Transport proteins: proteins which recognize a substrate and catalyze its movement across a membrane.

For facilitated diffusion, solutes move down their concentration gradient DG is negative because diffusion is energetically favorable.

XX

X

X

X

X

XX

X

X

X

X

X

XX

X

X

X

XX

X

X

X

XX

X

X

X

X

X

XX

X

X

X

XX

X

X

X

X

XX

X

X

X

X

X

XX

X

X

X

X

Active transport: energy requiring reactions moving them against their gradients.

Ions are frequent targets of transporters.

Transport Across Membranes

X X

ATP

ADP

Simple Diffusion Across Membranes

Simple diffusion is always energetically favorable-- no cellular energy required because it is always a decrease in DG.

Diffusion rate is directly proportional to the difference in concentration.

Facilitated diffusion is enzyme mediate d- follows Michaelis-Menten kinetics and will plateau at the transporter’s maximum rate.

facilitated diffusion

Concentration of solute

tran

spor

t rat

e simple diffusion

Review Question #1

For neurons to bring more potassium ions (K+) inside the cell than outside, which type of transport is most likely to be used?

A. Simple diffusion

B. Facilitated diffusion

C. Active transport

D. None of the above

Review Question #2

For a steroid hormone such as testosterone, which of the following ways would be used for the hormone to enter a neuron?

A. Simple diffusion

B. Facilitated diffusion

C. Active transport

D. None of the above

Facilitated Diffusion

Transporters can move either 1 or 2 types of solutes at a time.

Uniport: transports 1 specific solute.

Cotransport: transports 2 different solutes at the same time (coupled) functionally, it requires both solutes so if 1 is absent, transport fails.

Symport: two solutes, same direction.

a

a a

bb

Antiport: two solutes moved in the opposite direction.

Facilitated DiffusionErythrocyte anion exchange protein: antiport protein facilitates the exchange of bicarbonate ions HCO3

- for chloride Cl-

Very selective and specific: 1 chloride, 1 bicarbonate, no other ions, both must be present to transport.

Antiport is required to overcome the electical work of transporting a single ion across the membran.e

Erythrocytes have the enzyme carbonic anhydrase to convert CO2 into bicarbonate HCO3

- goes from a membrane permeable molecule to an impermeable form.

Required to get CO2 from tissues to lungs; in lungs, the process is reversed. HCO3

-HCO3

-

HCO3-

HCO3-

CO2 + OH-

Cl-

Cl-

Cl-

Cl-

HCO3-

HCO3-

HCO3-

Facilitated DiffusionKCC2 (potassium-chloride cotransporter 2) is a symporter found on neurons in the nervous system.

Its job is to use the potassium electrochemical gradient to move chloride ions out of the cell (taking one potassium ion with it).

This is an absolutely essential protein for the maturation of neurons animals missing this protein die at birth.

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-K+

K+

K+

K+

K+

K+

K+

K+

K+

K+

K+

potential ~ -70 mV

return to case

Channel Proteins

Unlike transporters, channels form a hydrophilic corridor through a membrane to allow ions to move across a membrane directly; ie. no individual ion binding is necessary in a channel.

Channels are usually ion selective: allow movement of 1 or few ions.

Anion/cation selectivity is controlled by extracellular region (vestibule).

membrane regionextracellular

hole for ions

Channel Proteins

membrane

X

ligand gated voltage gated mechanosensitive

++

+

+

+

Ion channels are usually gated: closed until specifically opened usually only opened for a period of time before closing again.

Three broad types of channels:1. ligand gated: channels open in response to a chemical signal.2. voltage gated: channels open after changes in membrane potential.3. mechanosensitive: mechanical forces; i.e., touch and hearing (sound).

X

Review Question #3

For a neurotransmitter receptor such as glutamate, which type of transporter or channel do you think would be activated in order to use sodium ions to quickly depolarize the neuron (i.e., make the inside more positive)?

A. Uniporter

B. Symporter

C. Ligand gated channel

D. Voltage gated channel

E. Mechanosensitive channel

Review Question #4

In order to repolarize the neuron (i.e., take the neuron back more negative as quickly as it went positive), which type of transporter or channel is likely responsible if potassium is the ion out of the cell?

A. Uniporter

B. Symporter

C. Ligand gated channel

D. Voltage gated channel

E. Mechanosensitive channel

Review Question #5

What type of transporters or channels will restore the ion balances and move ions against their gradient?

A. Uniporter

B. Symporter

C. Ligand gated channel

D. Voltage gated channel

E. Mechanosensitive channel

return to case

Active TransportActive transport: energy requiring process to move a solute up a concentration gradient; must not only move the solute but couple it to an energy yielding reaction.

Three primary functions of active transport:

1. concentrates essential nutrients.

2. removes secretory or waste products from a cell.

3. maintains concentration of intracellular ions to keep a constant resting potential.

glycineATP

ADP

ATP

ADP

toxin

ATP

ADP

K+Na+

Active Transport

Direct active transport: accumulation of solute is coupled directly to an exergonic reaction, usually hydrolysis of ATP. Direct active transporters are often referred to as pumps.

There are four distinct types of ATPase pumps.

P-type ATPases: pumps themselves become phosphorylated hydrolysis of the phosphate provides –DG. Always cation transporters (+).

Best known example: Na+/K+ pump moving Na+ out and K+ in common in eukaryotes, less common (still present) in prokaryotes.

ATP

ADPP P

Na+

K+P

Active Transport

V-type ATPases: “vesicle”pumps force protons into organelles such as vacuoles, endosomes, and golgi complex.•Transport subunit is a transmembrane protein.•Peripheral protein component is the ATPase.•Allosteric changes in the peripheral protein are coupled to changes in the transport subunit that causes the actual movement of protons.

H+ H+

H+ H+

ATP ADP + P

F-type ATPases: “factor” multicomponent pumps superficially like V-type moves protons across a membrane, and composed of a transmembrane component and a peripheral ATPase component.

Found in mitochondria, chloroplasts, and bacteria.

Is reversible – proton gradients can force the synthesis of ATP.

Active Transport

F1 complex

F0 complex

matrix

H+

ADP +P ATP

Active Transport

ABC-type ATPases: “ATP binding cassette” large family of pumps from mostly bacteria, but eukaryotes as well.

Contains 4 subunits: 2 integral membrane proteins, 2 peripheral.

Generally different polypeptides associated in a complex, broad transport range.

Transporters carry ions, sugars, amino acids, or drugs, i.e., multi-drug resistance protein (MDR), or cystic fibrosis (CFTR).

ATP

ADP

Like all active transporters, ABC-typemove things AGAINST their gradient.

Active Transport

Indirect active transport: transport driven by ion gradients often associated with the simultaneous movement of other ions, usually Na+ or H+ down their concentration gradient.

Animal cells use sodium ion gradients to power uptake of many sugarsbacterial cells typically use proton (H+) gradients.

Cells also use indirect active transport to remove Ca2+ as an antiporter, i.e., Na+ ions come in while Ca2+ leave.

Some other mechanism creates thesodium or proton gradient.

Ca2+

Na+

Review Question #6

The protein which is damaged in cystic fibrosis is known as CFTR. It is a protein which uses ATP to move Cl- out of cells by binding to ATP, pushing the chloride out, hydrolyzing ATP to ADP + P and relaxing, thus releasing ADP. This is an example of a(n):

A. P-type ATPase

B. V-type ATPase

C. F-type ATPase

D. ABC-type ATPase

E. Indirect active transporter

Review Question #7

KCC2 is the potassium- chloride cotransporter mentioned earlier. It moves both Cl- and K+ ions out of the cell. This is a(n):

A. P-type ATPase

B. V-type ATPase

C. F-type ATPase

D. ABC-type ATPase

E. Indirect active transporter

Active TransportNa+/K+ pump is a key direct active transporter and is found in every cell cells keep sodium ions out and potassium ions in [Na+]out/[Na+]in ~ 22:1, while [K+]out/[K+]in ~ 0.03

P-type ATPase that is inherently directional; i.e., Na+ ions on the inside along with ATP binding, with K+ ions on the outside.

Main protein responsible for creating the sodium and potassium gradients in all cells, particularly useful in repolarizing neurons.

K+

ATP

ADP

Na+Na+

Na+ Na+Na+

Na+ P P

K+ K+

Na+ Na+Na+ Na+

Na+

K+ K+

Na+Na+Na+Na+

Na+ K+ K+K+K+

K+

K+

return to case

Transport Energetics

Just like every other chemical reaction, there is an overall -DG in every transport reaction (even in light driven ones, some energy is wasted).

2 different factors play a role in the energetics: concentration and charge.

For uncharged molecules, DG is determined only by concentration.

For the reaction Sout Sin

DG= RT*ln[S]in/[S]out i.e., if [S]in <[S]out, then -DG and spontaneous,

(Note that this is the same formula for any equilibrium reaction)

If [S]out< [S]in, energy is required to drive the solute into the cell.

Transport Energetics

For charged solutes, DG depends upon the electrochemcial gradient.

For the reaction Scout Sc

in

DG = RT*ln[Sc]in/[Sc]out + zFVp (added term to deal with the charge!)

R = gas constant 1.987 cal/(mol oK)T = temperature in degrees KelvinSc = charged solute, i.e., what ion is being consideredF = Faraday constant (23062 cal/mol) used with electricity in physicsz = charge on the ionVp = membrane potential in volts

i.e., negative charge with a negative membrane potential, DG goes up positive charge with a negative membrane potential, DG goes down.

Transport Energetics

What is the DG of Na+ ions moving into a cell at 25 oC if the resting Vp

is -60 mV, the internal [Na+]= 12mM and the external [Na+]=150 mM?

The chemical “reaction” for this transport is Na+out Na+

in

DG = RT*ln[Na+]in/[Na+]out +zFVp substitute into the equation...

DG = 1.987*298*ln(0.012/0.150) + (+1)*23062*(-.060)

DG= 592*ln(0.08)+ (-1383.72)

DG= -1495 - 1384

DG = -2879 cal/mol, so sodium ions flow into the cell (i.e., in the forward direction of the equilibrium “reaction”)

Review Question #8

For a negatively charged ion like chloride, if there is more chloride outside the cell than inside, how likely is it to move across the membrane at 25oC if the membrane potential is -70 mV?

Remember, DG = RT*ln[Sc]in/[Sc]out + zFVp

A. Likely – has a negative DG

B. Unlikely – has a positive DG

C. Could move either direction depending upon the concentrations

D. Can’t tell – not enough information given

Hint: Consider charge and concentration separately!

return to case