M&c Lectures 25-28

Front 1

What are 4 types of signaling?

Back 1

1. endocrine: involves large distances between signaling and target cells e.g. hormones released into circulation (pituitary)
2. paracrine: involves signaling molecules produced by one cell and that act on a neighboring cell (neurotransmitter)
3. autocrine: production and response to a signaling molecule by the same cell (e.g. growth factor in cancer)
4. cell contact-dependent: involves 2 cells that actually touch each other (Notch pathway)

Front 2

What can be a signaling molecule? How do they work?

Back 2

Signaling molecules can be proteins, peptides, amino-acids and their derivatives, lipids. What they have in common is that they do not cross the plasma membrane of the target cell. Exceptions include steroid hormones (whose receptors are inside the cell). Non-steroid signals require transduction of the signal from outside of the cell to inside, requiring cell surface receptors, whose extracellular portion of the receptor binds the ligand with high-affinity.

Front 3

What's in a G-protein coupled receptor?

Back 3

GPCRs are called G-protein coupled receptors because the C3 loop interacts with and activates G-proteins (heterotrimeric G-proteins)

G protein consists of α, β, γ subunits: β, γ always stick together; these two are associated with the α subunit that binds a GDP in its “resting” state (receptor unbound by ligand)

Front 4

Describe the steps of GPCR function

Back 4

1) Resting state : receptor unliganded, α subunit in GDP bound state bound to βγ
2) Ligand binds receptor → change in conformation of C3 loop → C3 binds α subunit of G-protein → stimulates guanine nucleotide exchange on α (GDP for GTP)
• GTP bound α has altered conformation
3) α-GTP dissociates from β/γ
4) α-GTP binds to a target or effector protein: effector only active when bound to α-GTP

RGS = Regulator of G protein Signaling
5) RGS binds α-GTP and helps it hydrolyze GTP → GDP; α-GDP now binds with β/γ; process starts over again


(intracellular GTP is MUCH higher than GDP; hence more available to take place of GDP in a switch)

Front 5

What is G-αs? What does it do?

Back 5

Is a G-protein subunit

activated by receptors for glucagon and epinephrine

binds Adenylyl cyclase; stimulating its activity

Front 6

Adenylyl cyclase description and effects

Back 6

o AC catalyzes the reaction: ATP → cAMP + PPi
• cAMP acts as a “second messenger” – low MW non-protein factors that activate intracellular signaling
o Has 12 transmembrane domains
• cAMP binds the regulatory domains of PKA and activates it

Front 7

PKA description and effects

Back 7

Protein Kinase A
• tetramer of 2 regulating R-subunits and 2 catalytic C-subunits
• Phosphorylates downstream targets

Front 8

What downregulates GTP, cAMP, phosphorylated protein levels along the GCPR -> cAMP pathway?

Back 8

o GTP hydrolysis by α and RGS
o cAMP phosphodiesterase: cAMP → AMP
• target of caffeine
o protein phosphatases remove phosphate from phosphoproteins

Front 9

What is Gq? What does it do?

Back 9

SP Serotonin receptors activate a G-protein w/ α-subunit Gq (as opposed to Gs in the glucagon pathway)
• Gq activates an enzyme called Phospholipase C β (PLC-β)

Front 10

PLC-β description and effects

Back 10

• PLC-β catalyzes the hydrolysis of PIP2 to yield DAG and IP3

Front 11

What do DAG and IP3 do?

Back 11

DAG and IP3 are BOTH second messengers
DAG binds and activates protein kinase C (PKC)
PKC has many substrates that regulate cell behavior

IP3 binds receptors on ER membrane
leads to the release of Ca2+ stores

Front 12

What is Gi? What does it do?

Back 12

An α-subunit called Gi
Gi inhibits adenylyl cyclase (opposite of Gs → cAMP)

Front 13

How does Cholera Toxin work?

Back 13

The pentameric part B of the toxin molecule binds to the surface of the intestinal epithelium cells. Part A detaches from the pentameric part upon binding, and gets inside the cell via receptor-mediated endocytosis. Once inside the cell, it permanently ribosylates the Gs alpha subunit of the heterotrimeric G protein resulting in constitutive cAMP production.
o adds an ADP-Ribose moiety to Arg 174 of Gs into GTP-bound state
o [cAMP] increases
This in turn leads to secretion of H2O, Na+, K+, Cl-, and HCO3- into the lumen of the small intestine resulting in rapid dehydration.

Front 14

How does Pertussis toxin work?

Back 14

Pertussis (whooping cough) Bordetella pertussis
• B.p. toxin ADP-ribosylates Gi → prevents Gi from binding receptor
o never activated
o [cAMP] increases

Front 15

Describe Receptor Tyrosine Kinases

Back 15

• Ligand includes insulin and epidermal growth factor
• Receptor auto-phosphorylates
Dimerization and autophosphorylation of receptors induced by ligand
• Autophosphorylation has 2 effects
o phos in catalytic region increases the specific activity of the tyr kinase
o phos poutside catalytic region generates phosphotyrosine residues that serve as binding sites for signaling proteins in the RTK pathway

Front 16

What recognizes the P-Y residues of RTKs?

Back 16

There are specific protein domains that specifically recognize phosphotyrosine w/in proteins at specific segment contexts (residues/protein environment)
• One domain that recognizes and binds phosphotyrosine is this way is called the SH2 domain – src homology 2 domain
• many proteins in RTK pathways have SH2 domains
• Two major types of SH2 domain containing proteins:
o Enzymes
• have SH2 domain and some enzymatic catalytic domain
o Adaptors
• have no enzymatic activity

Front 17

What binds RTK?

Back 17

PLC-γ and GRB2-SOS

What happens to each?

Hint 17

1. PLC-γ gets phosphorylated by RTK, its activity increases. By binding RTK PLC-γ is in proximity of its membrane substrate, PIP2. PLC-γ cleaves PIP2 into IP3 and DAG.

2. GRB2 (adaptor protein); has 2 SH3 domains, 1 SH2 domains; SH3 domain binds proline-rich SOS.
GRB2 brings SOS in proximity of Ras, facilitating SOS's GEF activity on Ras; activates Ras.

Front 18

Effects of RTK autophosphorylation

Back 18

1. in the catalytic region, phosphorylation increases the specific activity of the tyrosine kinase; continues to phosphorylate outside catalytic region
2. Outside the catalytic region, phosphorylation creates phosphotyrosines

How is a phosphotyrosine recognized?

Hint 18

Based on the context; i.e. surrounding residues

Front 19

Describe Ras and its actions

Back 19

Single protein, active when GDP bound; is tethered to membrane. RAS can hydrolyze GTP, but slowly.
Binds and activates Raf by allowing it to be phosphorylated by OTHER kinases.

What other factors affect the GTP/GDP binding states of RAS?

Hint 19

RGS = GAP of Ras - helps hydrolyze GTP

SOS = GEF of Ras

Front 20

Describe Raf and its actions

Back 20

• Raf is a cytoplasmic ser/thr kinase
o binds actives Ras
o When bound to Ras, Raf is phosphorylated by other kinase and activated
o Raf phosphorylates MEK

Front 21

Describe MEK and its actions

Back 21

• MEK is a dual specificity protein kinase (phosphorylates Y and T)
o MEK phosphorylates ERK

Front 22

Describe ERK and its actions

Back 22

• ERK is a Ser/Thr kinase that phosphorylates many substrates
o pERK phosphorylates cytoplasmic and nuclear substrates
o pERK can enter nucleus and phosphorylate transcription factors that changes gene expression

Front 23

Describe the entire RTK pathway

Back 23

Signal bind receptor; induces RTK dimerization and autophosphorylation; attracts PLC-γ (cleaves PIP2 -> IP3 + DAG) and GRB2-SOS (activates Ras through SOS mediated GEFing, which activates Ref, which phosphorylates MAPK, which phosphorylates ERK, which enters the nucleus and affects gene transcription)

Front 24

Describe the Notch pathway

Back 24

Notch Signaling
• Cell contact dependent signaling
• 2 cells must touch

• Notch = receptor
• Delta = ligand
Binding causes proteolysis of Notch
Intracellular region goes to nucleus
and binds other proteins and acts as part of a transcription factor complex

Receptor is also final effector

Front 25

What are carrier proteins?

Back 25

These operate in cycles, by binding solutes, undergoing conformational changes that move the solutes across the membrane, and returning to the initial conformation. As with conventional enzymes, each has a characteristic stoichiometry, Km, and Vmax, and is saturable.

Front 26

What are channels? Advantage over carrier proteins?

Back 26

Channels. These proteins allow for much higher rates of movement than the carrier proteins. They do not bind solutes or operate in cycles, but instead provide a pore through the membrane through which specific solutes can diffuse down their electrochemical gardients.

Front 27

What types of pumps are there?

Back 27

1. P-class
2. V/F class
3. ABC-class

Briefly, what differentiates them?

Hint 27

1. While all pumps have intrinsic ATPase activity, only P-class pumps become phosphorylated in the
course of a cycle.

2. These two classes are structurally similar to each other, and pump only protons. V-class
pumps contribute in the acidification of organelles such as lysosomes by pumping protons from the cytoplasm to the lumen of the organelle. F-class pumps, one type of which (F0-F1 ATPase) is highly expressed in mitochondria, typically run “in reverse”, using the movement of protons down their gradient to synthesize ATP.

3. These pumps bind ATP through conserved regions called ATP-binding cassettes (thus, “ABC”).
Unlike the P- and V/F classes, ABC pumps often transport uncharged and even hydrophobic molecules.

Front 28

Describe the function of P-class pumps, give an example of one.

Back 28

A prominent example of a P-class pump is the Na+/K+ ATPase, This pump removes 3 Na+ ions from the cell, and brings in 2 K+ ions, at the expense of one molecule of ATP. The key to its function is the presentation of Na+- and K+-binding sites with different affinities at the cytoplasmic and extracellular faces of the membrane.

Mechanism?

Hint 28

(1) In the initial pump conformation (E1), three Na+ ions occupy high-affinity sites (KD ! 0.6 mM) that are accessible from the cytoplasm. Also on the cytoplasmic side are two low-affinity sites for K+, which are unoccupied.
(2) ATP binds to the pump and is hydrolyzed by the pump’s ATPase activity, and a high-energy phosphate bond is formed with an aspartate residue on the cytoplasmic side of the pump.
(3) The high-energy phosphate bond then converts to a low-energy bond, releasing energy that is used to drive a conformational change to E2, a process called the “power stroke”. As a result, the Na+ ions move to low-affinity sites that are exposed to the extracellular space, where they are weakly bound. The E2 conformation also presents two high-affinity K+ binding sites to the extracellular side (KD ! 0.2 mM) .
(4) The three Na+ ions diffuse away from their low-affinity sites and into the extracellular space. At the same time, two K+ ions from the outside bind their high-affinity sites, causing hydrolysis of the aspartyl-phosphate bond.
(5) The loss of the phosphate group returns to pump to its E1 state, transferring the K+ ions to their low-affinity binding sites that face the inside of the cell, from which they dissociate into the cytoplasm.

Front 29

What is the effect of Na+/K+ ATPase function?

Back 29

The Na+/K+ ATPase establishes steep gradients for Na+ and K+ across the membrane, gradients that can be exploited to do work for the cell (K out/in = 150/4; Na out/in = 145/10). Due to the direct effect of the Na+/K+ ATPase, the intracellular concentration of Na+ is kept low, and intracellular K+ is kept high.
Note that the Na+/K+ ATPase is electrogenic: it expels three Na+ for every two K+ that enter the cell. This is an electrical current that produces a small voltage across the membrane, making the inside of the membrane a few millivolts negative to the outside. However, as we will see in the next lecture, cells can generate much larger voltages across the membrane by allowing Na+ and K+ to flow down their gradients through channels.

How is [Ca++] kept so low in the cell?

Hint 29

SERCA pumps in the sarcoplasmic reticulum: Relatively good structural data are available for SERCA, indicating that it contains 10 transmembrane helices, which provide binding pockets for Ca2+. According to a current model of SERCA operation, in the E1 state a high-affinity Ca2+-binding site (KD ! 1 x 10-7 M) is accessible to the cytoplasm. When ATP is hydrolyzed and a high-energy phosphate bond formed with an aspartate on the cytoplasmic side, a conformational change to E2 closes off the pocket from the cytoplasm, with Ca2+ trapped within. A subsequent series of transitional states, analogous to those described above for the Na+/K+ ATPase, transfers the Ca2+ to a low-affinity site that is exposed to the lumen of the sarcoplasmic reticulum. After Ca2+ diffuses from this site, the SERCA pump returns to its basal conformation. This activity keeps the cytoplasmic concentration of Ca2+ below 1 "M in most cells.

Front 30

Describe 2 ABC-class Pumps

Back 30

1. Multi-drug resistance (MDR) proteins are highly expressed in the epithelial cells of the intestine and kidney. They transport small, polar molecules, including some products of normal metabolism, but they can also pump a wide variety of drugs out of cells. Thus, tumors that overexpress MDR proteins are resistant to treatment by multiple and unrelated anticancer drugs.

2. The cystic fibrosis transmembrane regulator (CFTR) is expressed in the lung and other organs. Although structurally an ABC-class pump, it has no known “pumping” function. However, it incorporates a channel that is permeable to Cl-, and that is regulated by protein kinase A. Cystic fibrosis has been linked to loss-of-function mutations in CTFR, which reduce Cl- transport across pulmonary epithelial cells. As a result, the mucus secreted by these cells becomes abnormally thick, compromising gas exchange and predisposing the lung to infection.

What does ABC stand for again?

Hint 30

ATP-binding cassettes

Front 31

What are transporters?

Back 31

Transporters, like pumps, bind solutes and undergo conformational changes to ferry them across the membrane. In contrast to the pumps, the transporters have no ATPase activity; rather, they rely on existing gradients to move solutes.

What types of transporters are there?

Hint 31

1. Uniporters
2. Co-transporters
a. symporters
b. anti-transporters (exchangers)

Front 32

Define uniporter; provide an example. How does it work?

Back 32

Uniporters conduct a single species of molecule down its gradient, facilitating a process that is already thermodynamically favorable by circumventing the hydrophobic membrane barrier (facilitated diffusion).

Glucose uniporters (GLUT proteins) bind a single molecule of glucose at a time. A conformational change exposes the glucose-binding site alternately to the extracellular and intracellular sides, and the rate of cycling is accelerated by occupation of the binding site in either conformation (Fig. 3). In most cells, the concentration of glucose is higher outside the cell than inside, so the extracellular binding site is more likely to become occupied (this is the situation shown in Fig. 3). In such cells, the intracellular concentration of glucose is kept low by the rapid phosphorylation of glucose to glucose-6-phosphate. Following the conformational change, when the glucose is exposed to the low concentration inside the cell, it diffuses away from the binding site. With time, the GLUT protein returns to its initial conformation, and another molecule of glucose can bind from the outside.

Is it only one way?

Hint 32

NO. In some cells, the inside concentration of glucose is greater than the outside concentration, and the uniporter acts in reverse. For example, in the epithelial cells that absorb glucose from the intestine, the intracellular concentration of glucose often exceeds the concentration in the interstitial space, and a GLUT protein transports glucose out of the cell and into the interstitium. The movement of glucose through the cell, entering at one area of the membrane and exiting at another, is an example of transcellular transport

Front 33

Define antiporter; provide an example. How does it work?

Back 33

couple the thermodynamically favorable movement of one type of molecule (down its gradient) to the unfavorable movement of another (secondary active transport). Usually, it is the potential energy of the Na+ gradient that is tapped, with the entry of Na+ used to move some other solute against its gradient. Exchangers (also called antiporters) move the different solutes in opposite directions.

The Na+/Ca2+ exchanger is an antiporter that couples Na+ entry to Ca2+ efflux. Thus, it serves a role in Ca2+ handling that is complementary to the sequestration performed by the SERCA pump, and in cardiac cells it contributes to Ca2+ clearance more than SERCA does. It has a stoichiometry of 3 Na+ : 1 Ca2+, so it is electrogenic, accumulating positive charges inside the cell.

What would happen if I were to administer Digitalis to cardiac cells?

Hint 33

Digitalis inhibits the Na+/K+ pump; therefore the Na+/Ca2+ exchanger would lose the Na+ gradient needed to pump out Ca2+. Ca2+ levels rise; muscle contractility increases.

Front 34

Define symporter; provide an example. How does it work?

Back 34

Symporter couples the transport of a solute down its gradient with the movement of another solute up its gradient; in the same direction as the first solute.

Na+/glucose transporters (SGLTs) are widely expressed symporters that couple Na+ influx to glucose uptake (Fig. 5). The kidney expresses two types of SGLTs, which nicely illustrate the importance of stoichiometry in determining the effectiveness of a transporter in moving substances against their gradients.

What are the two Na+/glucose symporters in the renal tubule? How do they differ?

Hint 34

SGLT2, expressed in the apical (lumen facing) membrane early in the tubule (higher [glucose]) has a 1:1 Na:Gluc ratio.

SGLT1 has a 2:1 Na:Glucose ratio; can transport Glucose towards 3*10^4 times more concentrated cytoplasm.

Their combined effects essentially removes all the glucose from the urine

Front 35

What is the Nernst equation?

Back 35

Veq(S)= RT/zsF •(ln[S]o/[S]i)

where R is the gas constant, T is temperature in °K, zS is the valence of the solute S, F is Faraday’s constant, [S] !is the outside concentration of S, and [S] is the inside concentration of S.

At T = 20C, RT/F = 25mV, 2.3 natural log conversion factor; equation becomes:
Veq(S) = 58/z * log ([S]o/[S]in)

What does solving it for K (o/i = 4/150) and Na (150/10) yield?

Hint 35

V(k) = -91mV

V(Na( = 67mV

Front 36

What is a reversal potential?

Back 36

The value of Vm where the concentration gradient and the electrostatic force are balanced is called the equilibrium potential or the reversal potential (Veq, or Vrev). The reversal potential for K+ is called VK, for Na+ it is VNa, and so on.

What are V(K) and V(Na)

Hint 36

V(k) = -91mV

V(Na( = 67mV

Front 37

What is the driving force for an ion?

Back 37

Driving Force = Vm – Veq

What happens to K ions if the membrane potential is less than -91mV?

Hint 37

They go back into the cell (driving force is negative for K)

Front 38

Define conductance and resistance.

Back 38

The concept “ease of passing through the membrane” has an electrical correlate: conductance, which is the ability of a material to carry a current. Conductance is expressed in units of siemens; in electrophysiology, the picosiemen (or pS; 10-12 siemens) is usually used. The inverse of conductance is resistance, which has the ohm as its unit. Biological membranes have high resistance, and the total membrane resistance of a cell usually is expressed in megohms (M!; 10^6 ohms).

What is Ohm's law?

Hint 38

I = gV

alternatively, V = IR

Front 39

What is displayed on an I-V plot?

Back 39

Current (I) vs Driving Force (Vm - Veq)
What does the slope signify?

Hint 39

Slope is g, conductance (according to the formula, I = gV, g = I / V)

Front 40

Relationship between change in voltage and the conductance of a membrane

Back 40

the change in voltage is inversely proportional to the conductance of the membrane: V = I / g

the greater the resistance of the membrane to electrical current (e.g., the fewer open ion channels), the more the membrane potential changes in response to the application of a given current.

Front 41

Why don't cells exhibit instantaneous changes in membrane potential proportional to induced currents?

Back 41

Because they have capacitance-- the ability to store charges.
As the capacity of the membrane to store charge begins to saturate, more of the current is available for establishing a voltage difference across the membrane. If the applied current is sustained for long enough for Vm to reach a new steady state, this value is as predicted by Ohm’s law.

Front 42

What are rectifiers?

Back 42

Close upon depolarization

Rectifiers are electrical devices that permit current to travel in only one direction. This name was
applied to the Kir channels because, under experimental conditions, they were shown to carry large inward currents much better than large outward currents. However, the name can be somewhat confusing, because under physiological conditions K+ currents are always outward. In excitable cells, inward rectifiers often provide an outward current to keep the membrane polarized under resting conditions, but shut down upon strong depolarization so as not to resist the action potentials that are essential for the function of many excitable cells. We will elaborate on this property of inward rectifiers when we discuss the cardiac action potential in a later lecture.

What distinguishes different Kir channels?

Hint 42

Kir channels differ with respect to the extent that they discriminate against large outward currents. Strong rectifiers may become almost completely blocked when Vm is more than a few millivolts positive to Vm, whereas weak rectifiers retain substantial conductance at depolarized potentials.

Front 43

What's the difference between voltage gated channels and inward rectifiers?

Back 43

VGC's open upon depolarization; inward rectifiers close.

What are two types of VGCs?

Hint 43

Voltage-gated K+ channels

and

Voltage-gated Na+ and Ca2+ channels

Front 44

Describe the different parts of a Voltage-gated K+ channel...?

Back 44

Tetramers of six transmembrane helice (S1 - S6) proteins.

The most carboxy-terminal of these, S5 and S6, together with their linker region, are highly homologous to M1 and M2 of the inward rectifiers. This region has the same inverted tepee structure, the P-loop, and the characteristic K+ channel sequence in the selectivity filter. The helices that line the pore are S6. The four other transmembrane helices are largely embedded in the lipid bilayer. S4 is unique in containing multiple positively charged residues.

What does each do?

Hint 44

The S4 helices, which contain positively-charged residues, comprise the voltage sensor of the
channel. When Vm is well polarized, S4 is positioned near the intracellular side of the membrane, since it is attracted to the uncompensated negative charges in the cytoplasm (Fig. 7). With the S4 helices in this position, the S6 helices are pinched together near the intracellular entrance to the pore, preventing ion flow. This is the activation gate, which must be opened before the channel can pass a current.

Front 45

Describe the action of Voltage-gated K+ channels..?

Back 45

1. Closed when membrane is polarized.
2. Upon depolarization, all four S4 (+) regions moves to outer leaflet, opening S6 activation gate allowing K+ efflux.
3. Some K+ channels inactivate soon after opening through a ball (Lysine rich N terminus) occluding the pore
4. Inactivation gate opens upon membrane repolarization

What's the structural difference between K+ VGCs and Na+ or Ca2+ VGCs?

Hint 45

Instead of four subunits, the Na+ and Ca2+ channels are formed from a single polypeptide with 24 transmembrane helices that are arranged in four domains, each with six helices. These domains, numbered I – IV, are homologous with the K+ channel subunits.

Front 46

Describe the actions of Voltage-gated Na+ and Ca2+ channels...?

Back 46

1. Depolarization causes pore to open, causing influx of Na+ or Ca2+.
2. Channel inactivates; caused by positively charged residues on long intracellular loops occluding the pore.

Front 47

Describe Bartter's disease, the proteins involved, their normal function

Back 47

Bartter’s disease is characterized by excessive loss of NaCl, is caused by defects in proteins in the Thick Ascending Limb of the Loop of Henle.

Proteins involved include NKCC2 (symporter), ClC-Kb (Cl- channel), ROMK (K+ rectifier), and an Na+/K+ ATPase.

NKCC2 uses bringing Na+ down its concentration gradient to symport Cl- and K+ into the cell.

ROMK insures K+ is recycled into the lumen.

ClC-Kb keeps intracellular [Cl-] lower than the lumen

What happens with defects in each of these?

Hint 47

Defects in ROMK prevent K+ recycling; this lowers lumenal K+ that's needed for proper symport of Cl-, Na+ from the lumen through NKCC2.

Defects in ClC-Kb results in higher intracellular [Cl-], making NKCC2 function harder.

Defects in NKCC2 might prevent transport of all 3 ions from the lumen.

Front 48

What is the current clamp method?

Back 48

Method by which a cell is impaled with a microelectrode -- a glass capillary that has been heated and pulled to a fine tip with a tiny opening, and then filled with a salt solution. In addition to measuring Vm, the electrode can be used to depolarize or hyperpolarize the membrane by passing a current into the cell. Using Ohms Law (V = IR; V =I/g, g = I/V), the change in membrane potential can be predicted (when taking into account capacitance - i.e. why its not linear).

Why is it bad for measuring membrane conductances?

Hint 48

The current-clamp method is not generally useful for acquiring such data, because it provides poor control over the membrane potential or the currents that flow through the membrane. For example, if a drug blocks K+ channels that are open in the resting membrane, Vm will become less negative, which could close inward rectifiers and/or open voltage-gated channels. Consequently, neither Vm nor the current through the membrane (Im) is controlled, and we cannot solve for g = I / V.

Front 49

What is the voltage clamp method?

Back 49

In the basic voltage-clamp configuration , two microelectrodes are inserted into the cell. One of these electrodes serves to measure Vm. This voltage is fed into a differential amplifier, which compares it with a user-set command voltage that specifies the value at which Vm is to be held constant (i.e., clamped). The difference between these voltages is converted to a current, which is passed into the cell through the other electrode to keep Vm at the command voltage. In this way, negative feedback is used to keep Vm constant despite variations in the conductances of voltage- dependent channels, and the current that is injected by the amplifier is equal and opposite to the current passing through the membrane at all times.

How to calculate conductance using the voltage clamp method?

Hint 49

When the experimenter changes the command voltage (typically by producing an abrupt voltage step), the amplifier instantly passes whatever current is necessary to set Vm at the new command voltage. Since the values of Im and Vm are both known, the membrane conductance can be calculated from g = I / V

The voltage-clamp thus enables the active properties of the membrane, which change with Vm and time, to be analyzed.

Front 50

What do positive and negative current values mean with respect to the movement of positive cations?

Back 50

By definition, outward currents have positive values, and inward currents have negative values.

Given Veq(K+) = -91mV, what will I(K+) be when Vm = less than, more than, or equal to -91mV?

Hint 50

Because driving force is Vm - Veq, a Vm less than -91mV will yield a negative driving force. This means K+ will move in. Similarly, a value greater than -91mV will yield a positive value. This means K+ will move out, as is what happens during depolarization. When driving force is 0, (Vm = Veq), then K+ ions will have 0 net movement.

Front 51

What happens to the I-V graph of a cell when half of its K+ leak channels are blocked? If you doubled the leak channels?

Back 51

When half the channels are blocked, the conductance (slope) of the membrane is reduced by half. When the number of channels is doubled, the conductance (and the slope) doubles.

What happens to Veq(K+)?

Hint 51

It remains the same at -91mV

Front 52

What are the differences between the I-V graphs of voltage-dependent K+ channels, inward rectifiers, and K+ leak channels?

Back 52

Leak channels are linear; voltage-gated channels and inward rectifier plots change slopes dramatically at the Veq(K+).

What's the difference between the plots of K+ inward rectifiers and voltage-gated K+ channels?

Hint 52

While increasing Vm; at Veq(K+), inward rectifiers close (slope, conductance goes to ~zero) and voltage-gated K+ channels open (slope, conductance dramatically increases)

Front 53

What does the I-V plot of VG Na+ channels look like?

Back 53

At Vm < -50mV, the membrane is polarized and Na+ channels are closed. At Vm > 50mV, the membrane depolarizes, and the Na+ channels open, allowing Na+ in (ions in, plot falls below X-axis, g negative). The slope shifts up at -30mV (current still negative) and becomes linear at maximal conductance (maximal g), continuing through Veq (Na+) = +45mV, at which point, ion flow reverses.

Where is the magnitude of conductance and current maximal?

Hint 53

At -30mV, where the slope turns up towards maximal conductance/maximal slope, g.

Front 54

Will the predicted depolarization of a membrane be larger or smaller than Ohm's law prediction if the membrane contains inward rectifiers?

Back 54

It will be a larger depolarization, as inward rectifiers will close, reducing conductance ( g = I/V ) and increasing Vm.

What's the nickname of voltage gated K+ channels?

Hint 54

Delayed rectifier.

Front 55

Which toxins block Na+ VGCs? K+ VGCs? Na+/K+ pumps?

Back 55

Tetrodotoxin (TTX) blocks Na+ channels

Tetraethyammonium blocks K+ channels

Describe the current vs time graphs of a membrane given each channel blocker

Hint 55

Under TTX: There is no inward flux of Na+ ions and the ions do not flow inward upon stepping the voltage up; instead they leave to match the step voltage.

Under TEA: The current spikes downwards (ions moving in) and slowly rises to 0; i.e., no net movement of ions. It does not go above 0 (no K efflux)

Front 56

What is resting Vm of cardiac muscle?

Back 56

-90mV

Describe the sequence of a cardiac action potential.

Hint 56

1) K+ Inward rectifiers at rest provide the dominant conductance; close upon depolarization (preventing K+ efflux!!)
2) Depolarization opens Na+ channels, causing rapid influx of Na+
3) Slight repolarization as channels inactivate;
4) Ca2+ channels are slowly opening at this point, causing Ca2+ efflux (slow, sustained plateau of ~10mV)
5) Very-slow kinetic K+ channels open and Ca2+ channels close, causing repolarization

Front 57

name two things that increase propagation distance of an action potential!

Back 57

Wider diameter axon

Better insulation (myelination)

What type of conduction is used in myelinated axons?

Hint 57

Saltatory conduction, jumping from node to node of Ranvier

Front 58

What is the patch clamp method?

Back 58

An electrolyte-filled glass pipette, not very sharp, is pressed against the outside the cell membrane, forming a tight, high electrical resistance seal with the bilayer. The area of membrane that is circumscribed by the pipette tip often contains a small number of channels, and sometimes a single channel. The ion concentrations inside the pipette often resemble those of the extracellular fluid; alternatively, ions may be present at very non-physiological concentrations for experimental reasons. The pipette solution is connected to a feedback amplifier that clamps Vm at the command potential, and measures the current needed to do this. The small number of channels in the patch and the high resistance of the seal make it possible to resolve currents that are carried by
individual channels.

What three ways can you use patch clamp? What's the difference in data?

Hint 58

cell attached - intracellular environment remains intact
excised - pipette pulled away from cell attached, seal patch to pipette
whole cell - patch ruptured: intracellular environment can be dialyzed; macroscopic measurements taken instead of individual channels
The whole-cell method measures macroscopic currents flowing through the entire cell membrane, and single-channel currents cannot be resolved.

Front 59

What is a unitary current?

Back 59

current of a single channel, denoted "i"

What is a unitary conductance? What is a unitary form of Ohm's law?

Hint 59

γ= i / V (where “V” = driving force)

Front 60

What behavior do individual channel openings and closings show?

Back 60

Stochastic-- i.e. probabalistic behavior

What equation gives the probability of this type of channel being open?

Hint 60

Po = total time in open configuration / total time of recording

Front 61

How to measure Veq from single channel experiments?

Back 61

Veq can be determined from single channel data by varying Vm, measuring the current when the channel is open, and constructing an I-V plot.

What does a leak channel show? A voltage gated channel?

Hint 61

Leak channel shows a linear relationship between i, V

voltage gated shows a linear relationship as well, indicating that the current is proportional to the driving force, and that conductance of the open channel is not voltage-dependent.

The rectification of voltage-gated conductances that is observed in macroscopic recordings reflects voltage-dependent changes in PO of many individual channels