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“To Be or Not to Be … an Inhibitory Neurotransmitter

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1 “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.”

2 record them chatting away.”
“You must need a small Microphone to record them chatting away.” “Not sound,” Jessica replied. “You record electrical activity with a small electrode stuck into the cell. Every time I stimulate them with neurotransmitter I get some spikes.” She flipped through a couple of open windows on her laptop. “Like this one here. It’s called a trace.” Image of neuron by MethoxyRoxy, CreativeCommons Attribution-ShareAlike 2.5 3 sec 40 mV

3 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 much sense to me. Aren’t axons and dendrites just like wires that connect to 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 Click to review neurons and ions.

4 “Wait a minute,” George said. “That doesn’t make sense. I thought
membranes were supposed to keep ions and stuff like that in cells.” “They do,” Jessica agreed, “but proteins can help molecules move through a membrane. Our cells have a lot of different proteins on their membranes, especially neurons. Some of us may even have more neurons than others.” Cl- Ca2+ Na+ Na+ Na+ Cl- Cl- Na+ K+ K+ Na+ Na+ Cl- Cl- Cl- Ca2+ K+ Ca2+ K+ K+ Cl- Na+ Cl- Cl- Cl- Cl- K+ Na+ Ca2+ Na+ K+ K+ Cl-

5 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.

6 “Electrochemical gradient? Sounds like chemistry to me,” George
said. “Are you telling me it works just like diffusion of a dye in water?” Jessica smiled. “Exactly! Except it’s not just concentration that moves the ions but also electrical charge. Neurons are normally negatively charged on the inside. They spike when they become more positive.” Click to review electrochemical gradient Click to review types of diffusion. “If you say so. Then I guess you cause neurons to spike by dumping a neurotransmitter onto the cells?” “Yep. The one I’m studying is known as GABA,” Jessica replied. “It usually makes neurons more negative and keeps them from firing.”

7 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 review protein channels. Click to review protein transporters.

8 Clicker Question #4 George looked skeptical. “OK, I get that a channel works like a gate to 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

9 George nodded. “I get it. When you put, what was it, GABA, on your
cells, channels open and let ions in. So what kind of ion does GABA let 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 more negative, which keeps them from spiking. In some of my cultures, it’s doing 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.”

10 adding glutamate adding GABA adding acetylcholine culture day 9
neurotransmitter additions adding glutamate adding GABA adding acetylcholine culture day 9 culture day 14 3 sec 40 mV

11 George whistled. “I see what you mean. Day 9 neurons are spiking
when 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 the exact same GABA channel is there at both ages. If the same protein is there then it should react the same way.”

12 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’t know 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 needle in a haystack. I need to graduate this May!” “Well, I’m not really a scientist, so I can’t help you. But there must be something different between them. Maybe the chloride doesn’t like the smell inside the young neurons?”

13 “Ions can’t smell, George,” Jessica responded.
“Well something must be stimulating those neurons. I still think the 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 you at 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 reaction such as ion flow across a membrane? A. Entropy B. Enthalpy C. Free energy D. Uncertainty principle E. Newton’s first law of motion

14 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. “It worked! 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 makes ions move in a particular direction across a membrane.” “OK,” George asked after a long pause, “so how does it do that?” Click to review DG equations

15 “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 to make you do the calculations. In words, free energy is determined by two things: the concentration of the ion on both sides of the membrane...” “Right, diffusion,” George interrupted. “Yes. Both concentration and the electrical properties of the cell and the ion. Here, let me show you.” Jessica pulled out her mobile phone and brought up a picture. DG = RT*ln[Sc]in/[Sc]out + zFVp

16 Jessica explained, “If GABA is working as an inhibitory transmitter,
Cl- inhibition excitation Cl- K+ -70 mV to -40 mV Na+ -70 mV to -80 mV Ca2+ Jessica explained, “If GABA is working as an inhibitory transmitter, the neurons should become more negative. This is what they are doing in the older neurons. If GABA is working to excite neurons, then chloride must be flowing out of the cell. Follow?” “Barely, but go ahead,” George said.

17 “So I thought about free energy and the equation. One way to make
chloride enter a cell is to lower the intracellular concentration of chloride when it get older. There are only a few proteins that can do that, so I started looking.” Jessica smiled. “Have you ever heard of a protein called KCC2?” George stared, waiting. “You don’t really expect an answer, do you?” Cl- GABA GABA Cl- KCC2 young neurons old neurons

18 “KCC2 is a symporter, and carries potassium and chloride ions out
of the cell. I don’t see any effects based on potassium, but if chloride is lower on the inside of the cell, GABA will open the same exact channel on the cell surface and chloride will flow into the cell instead of out of the cell. KCC2 is only found in older neurons. Here’s the situation. For free energy, we only worry about chloride.” -70 mV overall 10 mM Na+ 120 mM K+ 20 mM Cl- -70 mV overall 12 mM Na+ 115 mM K+ 8 mM Cl- extracellular 150 mM Na+ 2 mM K+ 150 mM Cl- KCC2 young neurons old neurons

19 DG= RT*ln([Clin]/[Clout]) + zFVp
DG= 1.987*310* ln (0.020/0.15) + (-1)*23062*(-.07) DG= 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 Cl- GABA

20 DG= RT*ln([Clin]/[Clout]) + zFVp
DG= 1.987*310* ln (0.008/0.15) + (-1)*23062*(-.07) DG= 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

21 George looked impressed. “So by lowering the chloride concentration
in older neurons, KCC2 turns GABA into an inhibitory transmitter. Is that useful for anything?” Jessica smiled happily. “It seems to be important for making synapses initially. If target cells don’t become excited, they don’t know that an axon is trying to make contact with them.” George grinned at Jessica. “Hey, I’m all for making contact. Are you busy later tonight?” Jessica grinned back. “Sorry, George, I’m an old neuron. It takes a lot more than that to get me excited!”


23 Neurons in Action 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? Image of neuron by MethoxyRoxy, CreativeCommons Attribution-ShareAlike 2.5

24 Transport Across Membranes
Ca2+ Cl- Na+ Na+ Na+ Cl- Cl- Na+ K+ K+ Na+ Na+ Cl- Cl- Cl- Ca2+ K+ Ca2+ K+ K+ Cl- Na+ Cl- Cl- Cl- Cl- K+ Ca2+ Na+ Na+ K+ K+ Cl- 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.

25 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+ Na+ Cl-

26 Neurons and Neurotransmitters
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!) Cl- inhibition excitation Cl- K+ -70 mV to -40 mV Na+ -70 mV to -80 mV Ca2+ return to the case

27 Why Molecules Move Across Membranes
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

28 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. X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X

29 Transport Across Membranes
Active transport: energy requiring reactions moving them against their gradients. Ions are frequent targets of transporters. ADP X X ATP

30 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. simple diffusion transport rate facilitated diffusion Concentration of solute

31 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

32 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

33 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 b b a a Antiport: two solutes moved in the opposite direction.

34 Facilitated Diffusion
Erythrocyte 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. Cl- HCO3- Cl- Cl- Cl- HCO3- HCO3- HCO3- HCO3- CO2 + OH- HCO3- HCO3-

35 Facilitated Diffusion
KCC2 (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). Cl- Cl- Cl- K+ K+ Cl- K+ Cl- K+ Cl- K+ K+ K+ Cl- Cl- potential ~ -70 mV Cl- K+ K+ K+ K+ Cl- Cl- Cl- This is an absolutely essential protein for the maturation of neurons animals missing this protein die at birth. return to case

36 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). hole for ions Images generated using rasmol from 2BG9.pdb file extracellular membrane region

37 Channel Proteins 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 X Images generated using rasmol from 2BG9.pdb file membrane + + + + + ligand gated voltage gated mechanosensitive

38 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

39 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

40 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

41 Active Transport Active 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. ADP glycine ATP ADP toxin ATP ADP Na+ K+ ATP

42 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. Na+ ADP P P P ATP K+

43 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. ATP ADP + P H+ H+ H+ H+

44 Active Transport 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. H+ F1 complex F0 complex matrix ADP +P ATP

45 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). Like all active transporters, ABC-type move things AGAINST their gradient. ADP ATP

46 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 sugars bacterial 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 the sodium or proton gradient. Ca2+ Na+

47 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

48 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

49 Active Transport Na+/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. Na+ Na+ Na+ Na+ Na+ K+ Na+ Na+ Na+ Na+ K+ K+ Na+ Na+ ATP Na+ Na+ Na+ Na+ P P K+ K+ K+ ADP K+ K+ K+ K+ Na+ K+ return to case

50 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.

51 Transport Energetics For charged solutes, DG depends upon the electrochemcial gradient. For the reaction Scout Scin DG = RT*ln[Sc]in/[Sc]out + zFVp (added term to deal with the charge!) R = gas constant cal/(mol oK) T = temperature in degrees Kelvin Sc = charged solute, i.e., what ion is being considered F = Faraday constant (23062 cal/mol) used with electricity in physics z = charge on the ion Vp = 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.

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

53 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

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