L17 The Phosphoinositide Signalling Pathway and Ca2+ functions and mechanisms

list the signal reception and processing pathways

• steroid hormones

• ligand gated ion channel/receptor operated channels

• cyclic AMP pathway

• phosphoinositide pathway

• tyrosine kinase pathway

what receptor activates both the cyclic AMP pathway and the phosphoinositide pathway

seven transmembrane spanning receptor

what is ATP converted into in the cyclic AMP pathway

cAMP

what is PIP2 converted into in the phosphoinositide pathway

IP3

what are cAMP, IP3, DAG known as

second messengers or intracellular messengers

whats the first messenger

chemical signal

what does IP3 generate

intracellular Ca2+ signal

what does the intracellular Ca2+ trigger

the endpoint function in the cell

elevation in cytosolic Ca2+ concentration…

transduces external signals (e.g. hormones, growth factors, neurotransmitters) into a response

give examples of responses from increased Ca2+

• ion permeability

• secretion

• contraction

• metabolism

• fertilisation

• DNA synthesis

• development

give an example of Ca2+ triggering an event

fertilisation -triggering of the fertilisation envelope

intracellular calcium ion concentration is kept… in cells

low

cells use the transfer of high energy phosphate bonds as their…

energy currency (ATP) -via phosphorylation

calcium phosphate is extremely…

insoluble

what happens if the calcium ion level is very high…

it will bind to the phosphate and precipitate which kills the cell

when the cell evolved to use high energy phosphate as an energy currency, the cell evolved mechanisms to keep…

cytosolic calcium ion concentration very low (selective pressure)

what is the average intracellular calcium ion concentration in a resting cell

~100nM

what is average extracellular calcium ion concentration

~2-3mM

what does a Calcium-ATPase pump do

• pumps calcium ions against conc gradient by hydrolysing ATP

• pumps Ca2+ out of the cell

what are the two types of calcium-ATPase pump

• Plasma membrane Ca-ATPase (PMCA)

• Sarcoplasmic/endoplasmic reticulum Ca-ATPase (SERCA)

what stores calcium ions

• ER

• SR

what does a Na/Ca exchanger do

• exchanges calcium ions that are removed from the cell with sodium ions moving into the cell

• driven by an inward sodium gradient

how is the inward sodium gradient maintained

by the sodium potassium ATP-ase pump

calcium ions can accumulate in…

mitochondria, lysosomes and secretory granules

which two families of proteins bind calcium

• buffers (act like a sponge)

• sensors (bind and become active which activates the downstream function of the cell- transduce Ca2+ signal into the endpoint functional response)

give an example of a buffering protein

parvalbumin

give an example of a sensor protein

calmodulin

what are OFF mechanisms

act to turn off the Ca signal

list the off mechanisms

• Ca-ATPase pumps

• Na/Ca exchanger

• mitochondria

• proteins (and lipids)

why is there not just one very effective Calcium off mechanism?

OFF mechanisms operate over different Ca2+ concentration ranges

order the OFF mechanisms from lowest to highest calcium concentrations

• endoplasmic reticulum

• plasma membrane Ca-ATPase

• mitochondria

• plasma membrane Na-Ca

as the calcium concentration increases in the cell…

the pumps begin to work really hard

calcium signals trigger what during normal development which is why OFF mechanisms work really hard when calcium levels increase- cell has to be careful to accidentally trigger other physiological events

cell death

• necrosis

• apoptosis

why do mitochondria and plasma membrane Na-Ca have high affinity to calcium concentration

protect the cell when calcium concentrations are dangerously high without impacting normal responses

what is the average calcium level in the SERCA

~500microM

InsP3 (IP3) acts on…

IP3 receptors (inositol 1,4,5-trisphosphate receptors)

• receptor

• calcium ion channels

• from ER to the cytosol

how is the IP3 generated

phosphoinositol signalling pathway

what do Ryanodine Receptors (RyRs) do

release calcium

what are the 3 main types of Ca2+ signal the cell wants to produce

• elementary events

• Global Ca2+ wave (intracellular)

• Global Ca2+ wave (intercellular)

what are elementary events

highly localised inside the cell

list examples of elementary events

• growth-cone migration

• membrane excitability

• mitochondrial metabolism

• vesicle secretion

• smooth muscle relaxation

• mitosis

• synaptic plasticity

what is a Global Ca2+ wave (intercellular)

wave isnt restricted to just one cell (e.g. wave moves across whole monolayer) -coordinated activity

list examples of Global Ca2+ waves (intracellular)

• fertilisation

• smooth muscle contraction

• skeletal muscle contraction

• cardiac muscle contraction

• liver metabolism

• gene transcription

• cell proliferation

list examples of Global Ca2+ wave (intercellular)

• wound healing

• ciliary beating

• glial cell function

• bile flow

• insulin secretion

• smooth muscle-induced nitric oxide synthesis in endothelium

describe the Phosphoinositide (PI) pathway

• chemical signal induces a conformational change in a 7 membrane spanning receptor

• the III cytoplasmic loop of the receptor activates a G protein (G_q)

• G_q stimulates an amplifier, PLC

• PLC acts on a minor membrane phospholipid PIP2 to give a DAG and IP3

• DAG acts within the plane of the lipid bilayer to stimulate its effector PKC

• IP3 diffuses into the cytosol where it acts on ion channels to release Ca2+ stored up in the endoplasmic reticulum

• the onset of the Ca2+ signal is augmented by a process of CICR

what does PLC acting on a minor membrane phospholipid phosphatidyl inositol 4, 5-bisphosphate (PIP2) to give a diacylglycerol (DAG) and inositol 1,4,5 trisphosphate (IP3) represent

a bifurcation in the signalling pathway because both DAG and IP3 are second messengers

what does PLC mean

phospholipase C

what does PIP2 mean

phosphatidyl inositol 4, 5-bisphosphate

what does DAG mean

diacylglycerol

what does IP3 mean

inositol 1,4,5 trisphosphate

what does PKC mean

protein kinase C

what does CICR mean

calcium induced calcium release

what are the 3 ways calcium can act after the PI pathway

• Ca2+ acts through a protein kinase

• Ca2+ uses specific binding proteins (calmodulin, troponin C) to induce contraction in muscle cells

• Ca2+ can act directly on ion channels to influence excitability

what is the amplifier (for the phosphoinositide pathway)

Phospholipase C-beta (PLC-beta)

what does DAG do

activates PKC

what does IP3 do

releases Ca2+ from the ER

what does PLC-beta activate

DAG and IP3

what does trisphosphate mean

3 phosphates on 3 different carbons

what does triphosphate mean

all phosphates on the same carbon