Part 2 (final exam)The phosphoinositide pathway and calcium ions- The phosphoinositide cascadeo 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 is secreted…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 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-β) is activated PLC-β 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 membraneo IP3 is a small, water soluble sugar phosphate; capable of rapid diffusion throughout the cytosolo 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 lumeno 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 kinaseC (PKC)o DAG is a lipid molecule that remains in the membrane after formationo 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 proteinso It has important roles in cell growth and differentiation, cell metabolism, transcription activation and the regulation of signaling pathwayso PKC, if unchecked or wrongly activated, can lead tocancer If one genetically engineers the overexpression of active PKC in micetumors in susceptible mice- Calcium ions as second messengerso Ca2+ ions bind to various target molecules, triggering specific responses, socalcium ions can also be considered to be intracellular messengerso 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 messengero 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 cytosolo Calcium ions are very different in structure from cyclic nucleotides, inositol phosphate and other second messengers They are not synthesized or degraded enzymaticallyo The Ca2+ concentration in a particular cell compartment is controlled by regulated activity of Ca2+ transporters and channels located in membranes surrounding that compartmento 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 cytosolo Na/Ca exchangers in plasma membrane also helpo 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 membraneo 2. Opening IP3 receptors via the phosphoinositide cascadeo 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 receptoro 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 muscleo E.g. Enhancement of DAG’s ability to activate protein kinase Co E.g. vesicle fusion and alteration of cytoskeletal 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
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