BIO304 - Midterm #2

What are voltage-gated calcium (CaV) channels?

What are voltage-gated calcium (CaV) channels?

Open when action potential reaches nerve terminal for calcium influx

What is the function of calcium influx via CaV channels?

Interact with proteins to release presynaptic neurotransmitters from vesicles

What is the function of calcium influx at NMJ?

Drives contraction of contractile filaments

What is the significance of calcium in neurosignalling?

Transforms inert electrical signal of sodium & potassium to cellular processes

What are 2 functions of cytoplasmic calcium?

Binds oxygen atoms + causes protein conformational changes for signaling or activating mechanical processes

Where does cytoplasmic calcium bind oxygen atoms?

Carboxyl & carbonyl groups on amino acids

Draw the Bohr diagram for a calcium atom.

Ca = 2, 8, 8, 2

Draw the Bohr diagram for a calcium ion.

Ca2+ = [2, 8, 8]^2+

What are 6 signaling / mechanical processes activated by cytoplasmic calcium?

Vesicle exocytosis, muscle contraction, activating other ion channels, changes in gene expression, apoptosis, intracellular signalling

What are 3 dangers of cytoplasmic calcium?

Precipitates phosphates (CaPO4) into crystals, triggers apoptosis, cannot be chemically altered for neutralization

What is phosphate precipitation as CaPO4?

Accumulates & becomes toxic

What are 2 sources of phosphates for CaPO4 precipitation?

DNA + ATP

What is the role of calcium in apoptosis?

Ca2+ regulatory checkpoint for programmed cell death, ex. during development

What is cytoplasmic [calcium]?

Very low levels, [Ca2+]in of 10^-4 mM <<< [Ca2+]out of 1 mM (10,000 fold difference)

What is cytoplasmic [calcium] vs. [potassium]?

Calcium 1.5 million fold less concentrated than potassium

What is the advantage of very low cytoplasmic [calcium]?

Calcium is a good transient cytoplasmic signaling molecule

What are 3 processes where cytoplasmic [calcium] increases transiently?

Neuronal excitation, muscle contraction, after a stroke by positive feedback

What are 3 components of calcium-sensitive fluorescent indicators (GCaMP) for optical recording?

GFP, Ca2+ sensor protein calmodulin (CaM), M13 alpha helix of muscle protein myosin light chain kinase

What is calmodulin (CaM)?

Conserved eukaryotic protein with four Ca2+ binding sites

What is fluorescence of GCaMP?

Increase Ca2+ levels = increased GCaMP fluorescence

• What are 5 steps in the mechanism of GCaMP?

(1) APs cause Ca2+ influx, (2) Ca2+ activates CaM, (3) CaM binds M13 helix, (4) M13 helix pulls on GFP for conformation change, (5) increased GFP fluorescence

What is the limitation of GCaMP?

Calcium influxes are long-lasting so don’t have temporal specificity to rapid action potential

• What are 6 steps in excitotoxicity after a stroke?

(1) Blood clot stops blood flow to brain region, (2) no oxygen & glucose inactivates sodium-potassium pump depolarizes Vm closer to AP threshold, (3) neurons produce barrage of AP releasing excitatory glutamate, (4) no oxygen & glucose inactivates glutamate transporters for reuptake, (5) postsynaptic neurons produce barrage of APs propagating glutamate flood, (6) excess calcium & zinc enter to trigger apoptosis

How does lack of oxygen & glucose depolarize Vm?

Decreased ATP = decreased sodium-potassium pump activity = abnormally depolarized RMP = higher frequency of APs

What causes neuronal death from a stroke?

Calcium-induced apoptosis rather than direct lack of oxygen & glucose

What is stroke treatment for blood clots?

Thrombolytics dissolve blood clots to restore blood flow (ex. tissue plasminogen activator, tPA)

What is stroke treatment for NaV channels?

Inhibitors of NaV channels reduce # of action potentials generated

What is stroke treatment for glutamate receptors?

Inhibitors of glutamate receptors reduce excessive stimulation

What is stroke treatment for calcium channels?

Inhibitors of calcium channels reduce intracellular calcium buildup

What are 2 ways of quickly removing calcium from cytoplasm?

Cytoplasmic chelators/buffers + pumps & exchangers

How do cytoplasmic chelators/buffers remove cytoplasmic calcium?

Bind free Ca2+ to remove from solution

How do pumps & exchanges remove cytoplasmic calcium?

Extrude Ca2+ from cytoplasm to cell exterior or intracellular components (sarco/endoplasmic reticulum, mitochondria)

What are calcium pumps?

Pump calcium out of cytoplasm

What are 2 types of calcium pumps?

Plasma membrane calcium ATPase (PMCA) + sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA)

What is the sarcoplasmic reticulum (SR)?

ER of muscle cells storing Ca2+ for secretion during muscle contracton

How are PMCA & SERCA related to sodium-potassium exchange pump?

P-type (phosphorylated intermediate)

What is the structure of PMCA & SERCA?

Alpha-subunit (N & C terminus inside) with no beta-subunit

What is PMCA?

Pumps out 1 Ca2+ ion out of cell per cycle via hydrolysis of single ATP molecule

What are 4 human PMCA alpha genes?

Alpha 1-4 (brain/ubiquitous, brain & muscle, brain & muscle, broad distribution)

What are human PMCA alpha1 mutants?

Lethal

What are human PMCA alpha 2 mutants?

Hearing loss & balance

What are human PMCA alpha 4 mutants?

Male infertility

What is PMCA in the human protein atlas?

ATPB1 to 4

What is SERCA?

Pumps out 2 Ca2+ ions into SR/ER per cycle via hydrolysis of single ATP molecule

What are 3 human SERCA alpha genes?

Alpha 1-3 (muscle contraction, muscle contraction & neurons, non skeletal muscle but expressed in cardiomyocytes)

What is SERCA in the human protein atlas?

ATP2A1 to 3

What is sodium-potassium pump alpha subunit in human protein atlas?

ATP1A1 to 4

What is sodium-potassium pump beta subunit in human protein atlas?

ATP1B1 to 4

What is the disadvantage of PMCA & SERCA?

Sluggish at removing Ca2+, can’t keep up with rapid Ca2+ influx from action potential

Where is SERCA highly expressed?

SR for efficient removal of cytoplasmic Ca2+ & restoration of SR Ca2+ stores

Where is PMCA sparsely expressed?

Cell membrane so only good at maintaining low cytoplasmic Ca2+ levels when neuron not highly active

What are calcium exchangers?

Remove cytoplasmic Ca2+ more efficiently than pumps

What is the mechanism of calcium exchangers?

Secondary active transport rather than ATP hydrolysis

What is secondary active transport?

Consume energy from existing [ion] gradients to move desired ions “uphill” against their gradients

What are 2 calcium ion exchangers?

NCX + NCKX

What are 2 names for NCX?

Na+/Ca2+ exchanger + sodium-calcium antiporter

What is NCX?

1 Ca2+ out for 3 Na+ in, thus can depolarize Vm

What is the abundance of NCX?

Most widely distributed sodium-calcium exchanger

Why can NCX operate in reverse?

Not electrically neutral as both Na+ & Ca2+ have inward gradient

What determines the direction of NCX?

Whether Na+ or Ca2+ experiences strongest inward pull (of greater amplitude)

What determines inward pull of Na+ & Ca2+ in NCX?

Net ion charge x driving force

What is equilibrium potential of sodium (ENa)?

+68 mV

What is equilibrium potential of calcium (ECa)?

+124 mV

What is the net charge of sodium for NCX?

+3

What is the net charge of calcium for NCX?

+2

When is Na+ pulled in & Ca2+ extruded by NCX?

| 3(Vm - ENa) | > | 2(Vm - ECa) | … near RMP (study lec 5-2 slide 19)

When is Ca2+ pulled in & Na+ extruded by NCX?

| 3(Vm - ENa) | < | 2(Vm - ECa) | … at depolarized potentials (study lec 5-2 slide 19)

What is NCX during a stroke?

Depolarization of Vm contributes to toxic Ca2+ influx

What are calcium exchangers as revolving doors?

Whichever ion pushes on door stronger gets in while other ion gets pushed out

What is NCKX (Na+/Ca2+/K+ exchanger)?

4 Na+ in & 1 K+ out for 1 Ca2+ out

What is the advantage of NCKX over NCX?

Removes cytosolic Ca2 more efficiently

What is the equation determining direction of NCKX?

| 4(Vm - ENa) - 1(Vm - EK) | vs. | 2(Vm - ECa) | … not electrically neutral just like NCX

What is the equilibrium potential for potassium (Ek)?

-90 mV

What is the direction of NCKX?

K+ & Na+ wins except at unrealistically high depolarization so no reversal

Why did NCX evolve if NCKX is more efficient at removing Ca2+?

NCX needs less energy so is effective most of the time besides rare stroke-like condition

What is the structure of NCX?

Transmembrane domain (9 transmembrane segments with N-terminus outside & C-terminus inside) + cytosolic domain (loop inside between 5th-6th segment)

What are 3 human NCX genes?

NCX1-3 (muscle, brain, brain)

What is NCX in the human protein atlas?

SLC8A1 to 3

What technology was used to solve structures of NCX proteins?

Cryo-EM > X-ray crystallography at solving structures of large proteins like pumps

What is AlphaFold?

Program that can predict protein structure from 1’ structure, verified experimentally

What is the structure of NCKX?

Transmembrane domain (11 transmembrane segments with N-terminus inside & C-terminus outside) + cytosolic domain (loop inside between 5th & 6th segment)

What is cleavage of NCKX?

N-terminal transmembrane segment cleaved

What are 5 human NCKX genes?

NCKX1-5 (retina, retina & brain, brain & smooth muscle, brain & smooth muscle, not expressed at membrane)

What is polymorphism of NCKX5?

Associated with white skin in individuals from Europe & Asia so might regulate Ca2+ in melanosomes

What is NCKX in the human protein atlas?

SLC24A1 to 5

What is gene duplication of calcium exchangers?

Gene in different cell types evolved specialized functions

What is [Cl-] in immature neurons & almost all other cells?

[Cl-]in ~ [Cl-]out

What is [Cl-] in mature neurons?

Actively extrude Cl- from cytoplasm so [Cl-]out >> [Cl-]in

What is the equilibrium potential of potassium (Ek)?

-74 mV

What is the equilibrium potential of sodium (ENa)?

+54mV

What is resting membrane potential (RMP)?

-60 mV

What is equilibrium potential of chloride (ECl) in immature neurons?

-5.6 (-6) mV with [100 mM]out vs. [80 mM]in

How does Cl- influence Vm in immature neurons?

Pulls Vm towards ENa by trying to depolarize Vm (more depolarized than Vm)

What is equilibrium potential of chloride (ECl) in mature neurons?

-74.9 mV (-75 mV) with [100 mM]out & [5 mM]in

How does Cl- influence Vm in mature neurons?

Pulls Vm towards Ek by trying to hyperpolarize Vm (more hyperpolarized than Vm)

What is the neurotransmitter glycine?

Excitatory neurotransmitter that activates postsynaptic Cl- channels (glycine receptors)

What was Kakazhu et al.’s study?

Developmental switch of Cl- channels in mice superior olive neurons

What was postnatal day 0 (P0) in Kakazu et al.?

Glycine caused depolarization of Vm

What was P15 in Kakazu et al.?

Glycine caused pronounced hyperpolarization of Vm that ceased action potentials

What was the Vm graph for P15 in Kakazu et. al?

Adding glycine ceased action potentials while removing glycine returned action potentials