Part 2 final exam The phosphoinositide pathway and calcium ions The phosphoinositide cascade o When the neurotransmitter acetylcholine binds to a smooth muscle cell and the smooth muscle cell contracts o or when a foreign antigen binds to a mast cell in the blood and histamine o the response to the stimulus is mediated by the release of calcium ions stored within the cell and by the activation of protein kinase protein kinase C PKC o The signaling pathway utilized in both cases is called the phosphinositide is secreted cascade It is based on the metabolism of a minor class of membrane phospholipids the phosphoinositides The phosphoinositides PI 4 P and PI 4 5 P2 are produced by the phosphorylation of phosphatidylinositol PI and PI 4 P As a result of a receptor protein GPCR activating a specific G protein Gq the target enzyme phospholipase C beta PLC is activated PLC catalyzes the hydrolysis splitting of the phospholipid PIP2 into 2 molecules both of which are second messengers in cell signaling 1 Inositol 1 4 5 triphosphate IP3 diffuses through the cytosol and releases Ca2 from the ER by binding and opening the IP3 gated Ca2 release channels in the ER membrane o IP3 is a small water soluble sugar phosphate capable of rapid diffusion throughout the cytosol o IP3 diffuses to smooth ER SER and binds to the IP3 receptor protein on the SER surface SER is a site of Ca2 storage in a variety of cells Ca2 pumps in the SER membrane pump into the lumen o IP3 receptor is a ligand gated Ca2 channel opened by IP3 IP3 receptor released Ca2 ions into cytosol from the lumen of the SER 2 Diacylglycerol DAG remains uncharged in the membrane and helps to activate the enzyme protein kinase C PKC o DAG is a lipid molecule that remains in the membrane after formation o PKC is a kinase that is recruited from the cytosol to the inner surface of the plasma membrane o PKC phosphorylates a wide variety of target o It has important roles in cell growth and differentiation cell metabolism transcription activation and the regulation of signaling pathways o PKC if unchecked or wrongly activated can lead to proteins cancer If one genetically engineers the overexpression of active PKC in mice tumors in susceptible mice Calcium ions as second messengers o Ca2 ions bind to various target molecules triggering specific responses so calcium ions can also be considered to be intracellular messengers o The effect of IP3 on the calcium concentration is usually transient since IP3 is rapidly broken down to IP2 by specific enzymes Effects of DAG PKC are generally longer lived Calcium as an intracellular messenger o Ca2 ions have a key role in a remarkable variety of cell activities Muscle contraction Cell division Secretion exocytosis Endocytosis Fertilization Synaptic transmission Metabolism Cell movement Calcium in the cytosol o Calcium ions are very different in structure from cyclic nucleotides inositol phosphate and other second messengers They are not synthesized or degraded enzymatically o The Ca2 concentration in a particular cell compartment is controlled by regulated activity of Ca2 transporters and channels located in membranes surrounding that compartment o Ca2 concentration in cytosol of resting cell is maintained at very low levels typically 10 7 M 0 1 micromolar The cytosolic level is about 10 000 times lower than that in the extracellular space It is very low because 1 The Ca2 ion channels of the plasma and SER membranes are normally kept closed making these membranes highly impermeable to this ion 2 The ATP driven Ca2 transport systems of the plasma and SER membranes pump calcium out of the cytosol o Na Ca exchangers in plasma membrane also help o Organelles also sequester Ca2 ions Ca2 is pumped out of the cytosol into the ER and mitochondria various molecules in the cytosol tightly bind the free Ca2 o The concentrations of Ca2 ions can be measured in a different part of living cells by injecting fluorescent Ca2 binding dyes into cells and monitoring the light they emit using computerized imaging techniques The intensity of dye fluorescence depends on the free Calcium ion concentrations Depending on the responding cell a particular stimulus such as a hormone or neurotransmitter may 1 Trigger a localized and transient increase of Ca2 ion concentrations in one part of the cell 2 Trigger a wave of Ca2 release that spreads from one end of a cell to the other 3 Induce sharp oscillations in free Ca2 ion concentration Mechanisms for elevating cytosolic calcium ion concentrations Stimuli trigger a sudden increase in cytoplasmic calcium ion concentrations by o 1 Opening Ca2 ion channels in the plasma membrane cascade o 2 Opening IP3 receptors via the phosphoinositide o 3 Activating another channel the ryanodine receptor on the SER membrane The ryanodine receptor is a calcium activated calcium channel It is activated when the cytosolic calcium concentration increases beyond a certain level As a result of its activation even more calcium ions pour into the cytosol from the SER lumen The receptor then rapidly inactivates and calcium entrance shuts off as rapidly as it began The result of Caclium activation of the ryanodine receptor o Suppose that an external stimulus causes a modest local entrance of Ca2 ions into the cytosol due to the activation of a plasma membrane calcium channel or an IP3 receptor o This local Ca2 increase triggers a large release of calcium from the SER through the ryanodine receptor resulting in a large spike increase in cytosolic Ca2 o These spikes may occur repetitively if the stimulus is maintained and they drive an enhanced response of proteins to Ca2 ions o The same basic mechanism creates the waves of Ca2 that can travel across the cytoplasm of cells from one end to the other Calcium ions affect the function of cellular proteins in a number of ways 1 Ca2 ions can directly activate or inhibit various enzymes no need for a kinase intermediate o E g activation of troponin in skeletal muscle o E g Enhancement of DAG s ability to activate o E g vesicle fusion and alteration of cytoskeletal protein kinase C structure function 2 Ca2 ions can directly activate open ion channels and change membrane potential 3 Ca2 ions can bind to and activate a protein called calmodulin o Ca2 calmodulin can then bind to and activate enzymes and channels including a potent kinase Ca2 calmodulin dependent protein kinase This can initiate kinase cascades that activate transcription factors o In this way a rise in
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