Channel Modulation1. Signals for modulation for the channels (specific K and Ca channels)a. Cytoplasmic second messengersb. Cofactorsc. Coupling proteinsd. Enzymes2. Properties of modulationa. Modulation follows the stimulus with a delayb. Modulation usually outlasts the duration of the stimulusi. Persistent intermediate messengersii. Covalent modifications of the channel3. Major stimuli inducing modulationa. Neurotransmitters – slow synaptic actionb. Hormones4. Effect of modulationa. Voltage dependence, availability and speed of gating of channelsb. Changes in subthreshold voltage trajectory, firing threshold, action potential shape of a cell5. Sympathetic effect on hearta. Norepinephrine is releasedb. cAMP as a second messengerc. cAMP stimulates phosphorylation of many proteins including L type Ca channelsd. Probability of opening of L type Ca channels is greatly increasede. Ca entering is thus greatly increased, increasing the muscle contraction6. Effect of modulation on Neuronsa. Decrease neurotransmitter releaseb. Depress fast synaptic transmissioni. Depress presynaptic release of Caii. Open presynaptic K channelsc. Potentiate transmitter releasei. Closing presynaptic or postsynaptic K channelsii. Thus presynaptic channel spike broadensiii. Postsynaptic cell is easier to fire7. Methods – gigaseal (A seal between a cell and a micropipette that has an electrical resistance in the order of a gigaohm)a. Whole-cell clampingb. Inside-out excised-path recording8. cAMP (cyclic AMP) – adenosine 3’,5’ – monophosphate [second messenger]a. adenylyl cyclase – synthesizes cAMP from ATPb. cAMP phosphodiesterase – breaks cAMP downc. causes protein phosphorylation as a way to change the activity of enzymesd. cAMP dependent protein kinase (PKA)i. protein activated by cAMPii. final player in most cAMP activated signaling pathwaysiii. phosphorylates specific serine and threonine residues9. G-proteinsa. GTP (Guanosine triphosphate) binding proteinsb. Heterotrimeric – Galpha, Gbeta and Ggamma subunitsc. Attached to plasma membrane by hydrophobic anchors d. In resting state carry GDP bound to their Galpha subunite. If they encounter agonist bound receptor, GDP is liberatedf. GTP is from cytoplasm takes its placeg. The G protein disassociates into two part – Galpha-GTP and Gbetagamma dimerh. Galpha-GTP associates with adenylyl cyclase which stimulates cAMP synthesisi. Eventually (Protein Kinase A) PKA is activatedj. Eventually GTP is cleaved to GDP in Galpha through GTPase activity10. Signal amplification of cAMPa. Agonist bound receptor can activate many G-proteins moleculesb. Adenylyl cyclase can stimulate synthesis of many cAMP moleculesc. Activated PKA can phosphorylate many serine and threonine