PSIO404 Exam 5

UofA, Dr. Price

5.1.1 Describe the common structural features of cation (Na+, K+, Ca2+) channel

5.1.1 Describe the common structural features of cation (Na+, K+, Ca2+) channel

1. they have a selectivity filter formed by P-loops that removes water from ions and permits passage of specific types of ions

2. they have a central cavity formed by multiple subunits that rehydrates ions that pass into it

3. they have a gate formed by the inner helices which is input-driven for opening/closing

5.1.2

1. Discuss the general structure of prototype 1 and prototype 2 cation channels, and

2. name an example for each type of channel.

prototype 1: transmembrane domains with one P-loop between S5 and S6. ex: voltage-operated cation channels

prototype 2: 2 transmembrane domains with one P-loop between S5 and S6. ex: ENaC, Kir, K2P

5.1.3 Compare the two general prototypes of cation channels with regard to number of subunits, voltage sensation capability, P-loops and subunit pore formation.

prototype 1: one subunit, 6 transmembrane domains, voltage sensor capabilities, one P loop

prototype 2: one subunit, 2 transmembrane domains, 1 or 2 P loops

5.1.4 Describe voltage gating’s basic theme (basic mechanism).

1. membrane depolarization

2. the S3-S4 paddle (voltage-sensitive helices 3b and 4) swing upward into the membrane

3. channel is opened

5.1.5 Briefly describe the structure of VOLTAGE-gated Na⁺ channels.

1 α subunit + 3 β subunits. α subunit forms the channel.

4 influx openings + 4 efflux openings

5.1.6 Briefly describe the function of VOLTAGE-gated Na⁺ channels.

1. propagate APs

2. inactivation: voltage gating causes refractory period to allow for unidirectional movement of APs

3. targets of local anesthetics and neurotoxins

5.1.7

1. Name and

2. very briefly describe the action of two of the four anesthetics or toxins which target the voltage-gated Na+ channel.

lidocaine: acts as local anesthetic

tetrodotoxin: highly toxic neurotoxin

action: both inhibit voltage-dependent Na+ channels to interrupt impulse propagation in the NS

5.1.8 Briefly describe the functions of ligand-gated Na⁺ channels (epithelial Na⁺ channels, or ENaCs). How do ENaCs act in concert with ion pumps and K⁺ channels to accomplish their function?

ENaCs regulate electrolyte balance across epithelia

ENaCs bring Na+ into the cell while K+ channels bring K+ out of the cell. this way, they regulate the amount of positive charge within the cell.

5.1.9 Briefly explain the role of ENaCs in the sensation of taste: in which one or two types of taste do ENaCs play an initiating role?

salty and sweet

5.1.10 Briefly discuss the structure of VOLTAGE-gated K⁺ channels.

prototype 1 structure with 4 unjoined α subunits that form a pore

each α subunit is associated with a β subunit which reversibly plugs the pore

5.1.11 Briefly discuss the function of VOLTAGE-gated K⁺ channels.

function primarily to terminate the AP (gated by depolarization)

5.1.12 Name and very briefly describe one of the two K_V channel blockers mention in lecture.

dendrotoxins: venom of mamba snake that blocks Kv channels at NMJ synapses

5.1.13 Compare and contrast the function of BK and SK channels.

BK channels: “big conductance K+ channels”, gated by depolarization and calcium, aid in relaxation of smooth muscle

SK channels: “small conductance K+ channels”, gated by Ca2+ alone, responsible for after-hyperpolarization phase of AP

5.1.14 Name and very briefly describe one of the two poisons/venoms mentioned in lecture which target BK and SK channels.

apamin (part of bee venom)

5.1.15 Discuss the basic structure and important physiological function of inwardly-rectifying K⁺ (K_ir) channels.

structure: have prototype 2 structure

function: open at negative membrane potentials and close upon depolarization, set resting membrane potentials and shape APs.

5.1.16 Describe the activation (gating) and function of GIRK channels (also known as K_ir3 channels).

gating: gated by Gβγ subunits of Gi proteins Gbg Gbg

function: the way inhibitory NTs cause hyperpolarization in the postsynaptic cell

5.1.17 Briefly describe one of the two examples of the operation of GIRK channels mentioned in class (Hint: both examples are effects of the autonomic nervous system, i.e. sympathetic or parasympathetic effects).

GIRKs exhibit parasympathetic actions on the heart

5.1.18 Discuss the structure, function, and critical physiological importance of K_ATP channels. Specifically, be able to explain how K_ATP channels enable beta cells of the pancreas to depolarize and thus release insulin when blood glucose levels are high.

structure: prototype 2 structure (Kir channel)

function: high glucose→high ATP→channel closes→depolarization→insulin release

importance: insulin regulation of blood glucose!

5.1.19 Describe the basic structure and function of K_2P channels, and give a couple of examples of how K_2P channels are used by our cells.

structure: prototype 2 structure (but NOT voltage-controlled)

function: control resting potential (background channels that don’t fully close upon depolarization)

examples of their use:

• mechanically-gated for osmotic pressure relief to prevent rupture

• temperature-gated for control of body temperature

• ligand-gated for transmission of pain sensations, protects neurons against overstimulation (stroke)

5.1.20 Explain how K_2P channels are employed by a cell 1) to regulate cell osmolarity, 2) to determine and respond to ↑ body temperature, and 3) to protect against neuron damage in the event of oxygen deprivation in the brain.

1. regulation of osmotic pressure (internal mechanical pressure): cell opens K+ and Cl- channels to move solutes out in an electroneutral way so cell function is not impaired and osmotic pressure is relieved.

2. body temperature control in hypothalamus: K2P channels close as temperature drops→depolarization→generates APs. changed neuron activity mediates regulation of body T.

3. regulation of blood supply to cells: arachidonic acid and lysophospholipids respond to O2 deprivation in the brain and prevent fatal accumulation of Ca2+ in brain cells

5.1.21 Briefly describe the general function of anion channels in the body.

inactivation: gating of a Cl- channel can hyperpolarize the cell

5.1.22 List the 3 families of chloride channels discussed in lecture.

1. ClC family

2. CFTR family

3. GABA & Gly receptor anion channels

5.1.23 Briefly discuss the structure and function of ClC channels, and be able to give 2 of the 4 specific physiological roles/examples for ClC channels mentioned in lecture.

structure: 2 subunits make a single 2-pore channel

functions: control of cell volume and osmolarity, electrolyte transport across the epithelia, pH regulation, acidification for bone resorption, setting the resting membrane potential, and inhibitory influences on neurotransmission

2 specific physiological roles:

1. ClC-K is important to endolymph production in the inner ear

2. ClC7 is used by osteoclasts to secrete HCl

5.1.24 Briefly discuss the structure and function of GABA and glycine receptor anion channels.

structure: composed of 5 subunits

function: Cl- influx causes hyperpolarization

5.1.25 List and briefly explain the effects of, two of the five types of agonists for GABA and glycine receptor anion channels given in lecture.

alcohol and benzodiazepenes are used as fear relievers

5.1.26 List one of the two industrial chemicals mentioned in lecture which are also antagonists for GABA & glycine receptor anion channels.

avermectin (an insecticide & NS stimulant)

5.1.27 Briefly explain the use of analeptics for the treatment of alcohol or barbiturate intoxication (not addiction, just intoxication).

antagonist for GABA and glycine receptor anion channels, reverses intoxication

5.2.1 Describe how [Ca2+] differs throughout the cytoplasm vs. within organelles vs. the extracellular space for a typical non-stimulated eukaryotic cell.

Cytoplasm: 0.1 µM = 0.0001 mM

Organelles: 1-10 mM

Extracellular space: 1 mM (4 orders of magnitude larger than cytoplasmic [Ca2+])

5.2.2 Explain how [Ca2+] in the cytosol is maintained by a cell.

Ca2+ pumps in the plasma membrane (PMCA and EX-Na), ER (SERCA), and mitochondria

• PMCA pumps Ca2+ out of the cell

• EX-Na pumps Ca2+ out of the cell (in exchange for Na+)

• SERCA allows for cytoplasmic Ca2+ uptake into the ER

5.2.3 Briefly discuss the function of LIGAND-gated calcium channels of the plasma membrane involved in calcium signaling.

gated by intercellular signaling molecules (i.e. arachidonic acid) or Ca2+ influx

the following signals activate these channels: hormones, NTs, cytokines, and environmental stimuli that activate PLA2

5.2.4 Discuss the role of PLA2 in calcium signaling across the plasma membrane.

PLA2 produces arachidonic acid, whch gates ARCCs (arachidonic acid regulated Ca2+ channels), which determines calcium wave frequency.

5.2.5 Briefly discuss the function of VOLTAGE-operated calcium channels (VOCCs) of the plasma membrane involved in calcium signaling.

gated by depolarization (APs)

connect changes in membrane potential with Ca2+-dependent cellular processes, like:

• secretion

• movement

• NT release at synapses

• muscle contraction.

5.2.6 Briefly discuss the function of PMCA and EXNa in the regulation of cytoplasmic [Ca2+] in calcium signaling.

PMCA and EXNa are Ca2+ pumps in the plasma membrane.

• PMCA: pumps Ca2+ out of the cell

• EXNa: pumps Ca2+ out of the cell in exchange for 3 Na+ ions. Has a depolarizing effect.

5.2.7 Name and give the ligands for the calcium channels of the ER.

1. InsP3R: InsP3 and S1P

2. RyR: cADPR (& possibly NAADP)

3. Ca2+ itself for 1 and 2 (calcium-induced calcium release)

5.2.8 What is cADPR and how does its formation link cell redox state of a cell to calcium signaling?

1. cADPR = cyclic ADP ribose

2. cADPR is formed from NAD+ by ADP-ribose cyclase, linking it to the NADP+/NAD+ ratio, which is a measure of cell redox state

5.2.9 Discuss in detail calcium-induced calcium release, and explain how the release of calcium is stopped.

Release: ER calcium channels (InsP3R and RYR) release calcium upon ligand binding, which gates other Ca2+ channels

Stop: these channels are regulated by Ca2+ in a biphasic mode—below 300nM causes positive feedback, above 300nM causes negative feedback.

5.2.10

1. Describe SERCA, and

2. explain it’s contribution to Ca2+ wave formation.

1. sarcoendoplasmic reticulum calcium-dependent ATPase

2. brings cytoplasmic calcium into the ER to maintain a long-term signal by the cell

5.2.11 Discuss intracellular calcium waves: formation, frequency, and how constant signals lead to depletion of Ca2+ from the ER.

Formation: ER channels and plasma membrane Ca2+ pumps are regulated biphasically to spike then rapidly lower cytoplasmic Ca2+.

Frequency: controlled by ARCC (arachidonic acid regulated Ca2+ channel)

• strong signals → higher Ca2+ wave frequency

Long-term Ca2+ signaling→ Ca2+ is slowly lost by the cell due to more Ca2+ being pumped out of the cell than can be recovered by the ER

5.2.12 Discuss in detail how Ca2+ stores are replenished during prolonged Ca2+ signaling.

1. Low [Ca2+]

2. association between STIM proteins and CRAC channels in the ER membrane

3. leads to interaction b/w ER and plasma membrane

4. Ca2+ entering the cell is immediately pumped into the ER by SERCA

5.2.13 Briefly discuss calmodulin’s (CaM’s) role in Ca2+ signaling.

CaM binds 4 calcium ions via EF-hand domains → changes conformation → activated CaM forms interactions with many signaling proteins

5.2.14 Discuss the unique structure of CaMKII and explain how it is able to sense calcium wave frequency in a cell.

Structure: composed of 12 subunits assembled into 2 rings of 6 subunits each.

Senses wave frequency: CAMKII is activated by Ca2+/CaM-dependent transautophosphorylation of subunits in a stepwise manner. The stronger the signal (increased calcium wave frequency) = more active CAMKII

5.2.15 Discuss how calcium controls the transcription factor NFAT.

calcium waves within a specific frequency range will trigger activation of NFAT

5.2.16 Describe NFAT signaling in Down Syndrome patients.

DSCR-1 inhibits calcineurin, resulting in decreased NFAT production and signaling.

DSCR-1 is a regulatory protein (in Down Syndrome) which causes underexpression of proteins involved in organ development/function and immune response.

5.2.17 Describe how calcium controls DREAM, and discuss how calcium plays a role in the transmission of pain signals.

DREAM is the only TF that Ca2+ interacts with. Ca2+ directly inhibits DREAM, decreasing pain transmission via spinal interneurons

5.2.18 Explain how and why Calpains are activated, and summarize their action.

Activation: At high frequency, Ca2+ waves eventually become a persistent elevated cytoplasmic Ca2+ concentration, activating Calpains

Action: apoptosis—cleave key elements in the apoptotic machinery like Bcl-2 family, caspase-12, etc.

5.4.1 Name the five families of small G-proteins.

Ras, Rho, Rab, Arf, Ran families

5.4.2 Briefly discuss each of the following for the Ras family of small G-proteins: localization, types of signaling it transduces, and activity regulation by GEFs and GAPs.

localization: activated → anchors to the inner side of the membrane

signaling types: mitogenic, differentiation-controlling, and pro-apoptotic signals

GEFs: activation

GAPs: inactivation

5.4.3 Briefly describe how arrestin-coupled signaling leads to Ras activation.

heptahelical transmembrane receptor phosphorylation → arrestin → Tyr kinase Src → adaptor Grb2, Shc → mSOS of Ras-GEF (activator) → Ras activation

5.4.4 Briefly describe how Tyr kinase-coupled signaling leads to Ras activation.

Tyr-kinase coupled R → adaptor Grb2, Shc → mSOS of Ras-GEF (activator) → Ras activation

5.4.5 Briefly describe how Calcium signaling leads to Ras activation.

ion channel-coupled R → Ca2+ → GRF and GRP of Ras-GEF (activator) → Ras activation

5.4.6 Briefly describe how GPCR signaling leads to Ras activation.

GPCR → PLC-B or PLCe → Ca2+ → GRF and GRP of Ras-GEF (activator) → Ras activation

5.4.7 List 4 major GEFs for Ras.

1. mSOS (Son of Sevenless)

2. GRF (Guanine nucleotide Releasing Factor)

3. GRP (Guanine nucleotide Releasing Protein)

4. PLCe

5.4.8 List 3 major GAPs for Ras.

1. p120 GAP

2. Neurofibromin

3. CAPRI

5.4.9 List 3 major Ras effectors.

1. p120 Ras-GAP

2. RAFs (Ras-Activated Factors)

3. PI3Ks

4. PLCe

5. GEFs of Ral proteins

5.4.10 Describe how RAF is involved in the mitogenesis module (MAP kinase module).

RAF is a MAP3K for the MAPK mitogenesis (ERK) module

ex: RAF→MEK→ERK

5.5.1 In brief, describe the overall difference between lamellipodia, filopodia, and actomyosin (stress) fibers.  Name the small G-protein which controls the development of each of the three structures.

lamellipodia: large broad extensions, controlled by Rac G protein

filopodia: long conical projections, controlled by Cdc42 G protein

actomyosin stress fibers: produce contractions, controlled by Rho G protein

5.5.2 List the three general steps of vesicular transport, and identify which step is controlled by Rab, by Arf, and by motor proteins.

1. vesicle budding + fission (Arf)

2. vesicle transport (motor proteins)

3. vesicle docking (Rab) + fusion (Ca2+)

5.5.3 In brief, discuss the critical role of coat proteins (COPs) in the process of vesicular transport.

concentrate transmembrane protein cargo in the area

5.5.4 In brief, discuss the role of tethering proteins and SNARE proteins in the process of vesicular fusion.

tethering proteins: tether vesicle to membrane

SNARE proteins: trigger fusion of vesicle with membrane

5.5.5 Explain the critical role of the protein synaptotagmin in vesicular fusion.

synaptotagmin is the Ca2+ activated protein that enables membrane fusion

5.5.6 Action potentials gate calcium ion channels at synaptic terminals.  With regard to synaptic transmission, briefly explain why it is a good design (upon calcium binding ) for SNARE proteins to not require energy input to perform vesicle fusion.

advantageous for efficient synaptic transmission (transmits signals quickly without wasting energy!)

energy is only required to unlink the SNARE proteins

5.5.7 For the process of vesicular formation (budding), briefly describe the actions (and regulation by input signals) of Arf.

Arf triggers vesicle formation

Arf-GEF stimulates vesicle formation

Arf-GAP → GTP hydrolysis → inactive Arf

5.5.8 In brief, describe the regulation of Rab by input signals, including the functions of both GDF and GDI.

GDI binds inactive Rab once it dissociates from the vesicle

GDF brings inactive Rab back to the vesicle to be reactivated by Rab-GEF

5.5.9 In brief, discuss the process of vesicular fusion.  Be sure to include the roles/actions of Rab, tethering proteins, synaptotagmin, SNARE proteins, GDI, GDF, and calcium.

1. recruitment of coat proteins & formation of vesicle by Arf

2. release of coat proteins & recruitment of tethering proteins like Rab

3. Rab → vesicle trafficking & tethering & docking

4. calcium → vesicle fusion

5. calcium activates synaptotagmin → membrane fusion

6. SNARE provides mechanical force required to fuse the vesicle with the membrane.

7. Rab proteins regulated by GDI and GDF which recycle them

5.5.10 Very briefly, describe the control of nuclear transport by Ran.  Identify the direction of protein translocation which requires the input of energy, and very briefly explain why.

Ran-GTP controls export of proteins into cytoplasm. requires GTP hydrolysis because it goes against the concentration gradient.

Ran-GDP controls import of proteins into nucleus