Cell Signalling.docx

Cell Signalling Signalling 1: Signalling through ion channels Ion channels are often selective for a particular ion. Ca2+ is a ubiquitous signalling ion. Concentration of calcium inside a cell can be determined by fluorescence. Distribution of ion in gradients Concentration gradients of ions can be...

Cell Signalling Signalling 1: Signalling through ion channels Ion channels are often selective for a particular ion. Ca2+ is a ubiquitous signalling ion. Concentration of calcium inside a cell can be determined by fluorescence. Distribution of ion in gradients Concentration gradients of ions can be exploited to transport molecules across phospholipid bilayer. There is a pH difference (proton gradient) between inside and outside of lysosomes. Ion transport can be passive or active. Passive ion transport is determined by the electrochemical gradient. Active transport can pump ions against their concentration gradient with the aid of ATP. How ion gradients are generated Ion gradients are generated by active transport (requires ATP) Na+/K+ concentration gradient can be generated by Na+/K+ pump which is a P-type ATPase. Both types of ions are actively transported. Stoichiometry is the number of ions the protein transports per ATP (e.g., for Na+/K+ pump it is 5) Phosphorylation provides the energy required for a conformational change. Ca2+ pumps generate Ca2+ concentration gradient across both plasma membrane. PM (plasma membrane) C(calcium)A (P-type ATPase) Ca2+ pumps generate Ca2+ gradients across intracellular stores. By a protein called SERCA S(sarcoplasmic) ER (endoplasmic reticulum) C(calcium) A (P-type ATPase) Ion gradients can be used to drive secondary transport. The Na+ gradient is used to drive Ca2+ efflux via the Na+ /Ca2+ exchanger (antiporter) NCX antiporter transports 3 Na+ ions along its concentration gradient, which provides energy for pumping 2 Ca2+ ions against its concentration gradient. Ion Channels Voltage–gated ion channels are evolutionarily related (i.e. they all have a common basic structure) (share a tetrameric structure) S1 is the voltage sensing domain, S5 and S6 combine to form the ‘pore domain’ where transport of ions takes place. Ion selectivity in K+ channels is mediated by the selectivity filter. This selectivity filter stabilises larger dehydrated K+ ions. Voltage sensing (S1) is mediated by positively charged residues in the S4 region. Regularly spaced arginine (R) and lysine (K) residues “sense” potential changes. Signalling 2: Electrical Signalling Membrane Potential The membrane potential arises due to ion gradients. Positive charges matched by “fixed anions” At time (T=x), a voltage difference of 4 is created by 2 positive ions moving out of the cell. Meanwhile leaving 2 fixed anions inside the cell, thus V=-4 as the cell is interiorly negative. At time of equilibrium (T=eq), K+ ions diffuse back into the cell down their electrical gradient as they are attracted by fixed anions inside the cell. However, some K+ ions also diffuse out down their chemical gradient, forming an equilibrium where V is the equilibrium potential and is a half of when T=x. At equilibrium electrical gradient and chemical gradient cancel out. The Nernst equation allows calculation of the equilibrium potential for a given ion. The equilibrium potential for K+ is -90 mV. This can be calculated: The equilibrium potential for Na+ is +60 mV following the same method. The membrane potential comprises charge imbalance of all permeable ion. The resting membrane potential arises primarily due to K+ imbalance. Most cells are more permeable to K+ than other ions (“leak channels”). Resting membrane potential is thus close to the equilibrium potential for K+ (-90 mV) The Goldman-Hodgkin-Katz equation allows calculation of the membrane potential. Permeability PK>>PCl>PNa Therefore effectively, we only need to consider the concentration difference of K+ ions. The Nernst equation is a simplified version of the GHK equation which allows calculation of the equilibrium potential for a given ion. Neurotransmission Ion gradients can be used for signalling purposes through opening of ion channels. Action potentials arise due to the concerted action of voltage gated Na+ and K+ channels. Voltage–gated Na+ channels depolarize the membrane. Permeability PNa>>PK>PCl (active) Voltage–gated Na+ channels depolarize the membrane from the equilibrium potential for K+ (-90 mV) to Na+ (60 mV) Voltage–gated K+ channels repolarize the membrane. Voltage–gated Ca2+ channels mediate neurotransmitter release at the synapse. Ligand-gated Ion Channels Ion gradients can be used for signalling purposes through opening of ion channels. Post synaptic ligand-gated ion channels respond to released neurotransmitter. Electrical signalling is enhanced at excitatory synapses. Electrical signalling is dampened at inhibitory synapses. Inotropic glutamate receptors The pore domain has an Inverted pore topology. They have 4 sub-units combining together to form the entire receptor. GABAA receptors Nicotinic acetyl choline receptors Neuromuscular Junction (NMJ) Activated nAChRs depolarize muscle cells. Depolarization initiates contraction E-C coupling Contraction involves Ca2+ release from the sarcoplasmic reticulum (SR) Signalling 3: GPCRs Signalling through receptors Receptors are present on the cell surface or within the cell There are two types of ‘switch’ mechanisms present on the surface of cells: Through Phosphorylation -Phosphate driving activation Through GTP-binding proteins -In off stage the receptor is bound with GDP, to activate the switch GDP is replaced with GTP. This requires ATP which yields the phosphate group required for activation. -Switching is indirectly associated with the protein There are two main types of receptors: GPCRs -activated by a variety of ligands like hormones (e.g. adrenaline), neurotransmitters (e.g. glutamate) and odorants. -However, many are ‘ORPHANS’. GPCRs share a common structure. They have 7 transmembrane regions. GPCRs couple to heterotrimeric G-protein In the inactive stage, GDP is bound to the alpha subunit of the G protein. When signalling molecule binds, G-proteins associates with GPCR. GDP is replaced by GTP. Beta and Gamma subunit is dissociated. Anything that promotes the GDP to GTP transition is called a GEF (Guanine Nucleotide Exchange Factors) Signalling is via both α and βγ subunit. Signalling involves second messengers. cAMP Activation of adenylyl cyclase produces cAMP. Regulation of messenger synthesis by G-protein α subunits αs subunits activate adenylyl cyclase. αi subunits inhibit adenylyl cyclase. Adenylyl cyclase is reciprocally regulated by distinct α subunit. Many effects of cAMP are mediated via protein kinase A Firstly, cAMP dissociate the Catalytic domain (C) from the Regulatory Domain (R). Then, the Catalytic domain (C) phosphorylates the substrate, thus activating the substrate. cAMP signals are terminated by phosphodiesterase (degrades cAMP to AMP with the help of one water molecule) cAMP and the fight-or-flight response. Adrenaline activates β-adrenergic receptors and the cAMP pathway in the heart. PKA phosphorylates several proteins which increase Ca2+ levels. Increased Ca2+ levels increase force of contraction. IP3 and DAG Activation of phospholipase C-β produces IP3 and DAG. The substrate, Pl4,5-bisphosphate is broken down into a hydrophilic head-group (IP3) and a hydrophobic tail group (DAG). αq subunits activate Phospholipase Cβ (In addition to s that activates a.c and i that inhibits a.c. ) IP3 activates intracellular ligand-gated ion channels. IP3 binds to IP3 receptors which releases calcium in the context of muscle contraction. IP3R receptors are present in all cells. Many effects of Ca2+ are mediated by calmodulin. Ca2+ binding induces conformational changes and binding to target proteins. The grey subunit is the Cam Kinase, which can be associated with calmodulin to activate the protein. Cam Kinase is inactivated, and calmodulin is activated by Ca2+ binding and associating with calmodulin. The protein then undergoes ‘auto-phosphorylation’ to become fully activated (with a phosphate group bound). Ca2+ signals are terminated by Ca2+ pumps and exchangers. PMCA and SERCA (delivers to sarcoplasmic and endoplasmic reticulum) removes Ca2+ from cells. Na+/Ca2+ exchangers drive calcium out of the cell using the concentration gradient of Na+ ions. Many effects of DAG are mediated by protein kinase C (which requires Ca2+ to be fully active) DAG and Ca2+ (released by the effect of IP3) conspire together to activate PKC. Enzyme-linked receptors These receptors are called receptor tyrosine kinases are activated by growth hormones. Receptor tyrosine kinases dimerize and autophosphorylate upon activation. PDGF receptors and FGF receptors are responsible for this. Activated receptor tyrosine kinases recruit other proteins. A classic example is Mitogen-activated protein kinase (MAP kinase) signalling. Grb-2 is a protein that recruits other proteins. Sos is recruited by Grb-2 and promotes the activation of the 3rd protein called Ras. Ras is a monomeric G-protein (inactive-GDP, active-GTP). A phosphorylation cascade (also called a protein kinase cascade) then happens where one signalling activates another and the activated molecule then goes on and activates another. At the end, mesoderm induction happens (transition from bilaminar to trilaminar disk).