Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Gating of M channels by PIP2Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36Slide 37Slide 38Slide 39Slide 40Slide 41Slide 42Slide 43Channel Modulation and Spatial DistributionChannel ModulationSignals from various membrane receptors alter the properties of channels, changing the responsiveness of cells and the computations they perform.The signals involve cytoplasmic second messengers, cofactors, interacting proteins and enzymes.Modulation follows the stimulus with a delay, and often outlasts the duration of the stimulus.Multiple channels are modulated in response to one agonist. Channel-protein complexes help maintain spatial and temporal specificity of channel modulation.Channel Modulation in Cardiac Pacemaker CellsAutonomic regulation of the heartbeat+PKA-ATP-adrenergicreceptorNAM2 muscarinicAch receptorAchGsadenylylcyclaseGiG GcAMPAMPPDEphosphatasechannelschannelsPSympatheticNorepinephrine (NA)ParasympatheticAcetylcholine (Ach)AP in pacemaker heart beatfight or flightAP in pacemakerheart beatrest and digestCardiacPacemakerL-type Ca2+ channelsIon channels in cardiac pacemakerK+ channelsdelayed rectifierinward rectifierhyperpolarization-activated current IhL-type Ca2+ channelsT-type Ca2+ channelsDiFrancesco D et al, 1986-adrenergic modulation of L-type Ca 2+ channelsBean et al.Delayed onsetlong-lasting effect+PKAATP-adrenergicreceptorNAGsadenylylcyclaseGcAMPAMPPDEphosphataseL-type Ca2+ channelsP-adrenergic modulation of L-type Ca 2+ channelscholeratoxin+forskolin+exogenouscAMPactivated PKAL-type Ca2+ channelspurified L-type Ca2+ channelsactivated PKAATPL-type Ca2+ channelsPreconstitute into lipid vesiclesPKA phosphorylation of L-type Ca 2+ channelsmeasure calcium influxcAMP modulation of L-type Ca2+ channelssingle channel recordingYue et al, 1990IA,tot = NA × (gA × ΔVA) × PA(open)AchM2 muscarinicAch receptor30-100 ms L-type Ca2+ channels inward rectifierK+ channelsecondsAch-modulation of ion channelsSoejima M and Noma A, 1984Inward rectifier K+ channels ‘clamp’ neuron at resting membrane potentialEMIM0EKGiGM2 muscarinicAch receptorAchVmGIRK: G-protein coupled inwardly rectifying K+ channelvoltage-dependentactivationmillisecondsPKA-dependentmodulationsecondsG-mediated modulationmembrane-delimited30-100 msG-mediated modulation: a membrane-delimited shortcutPKA-ATPmuscarinicAch receptorAchadenylylcyclaseGiGcAMPCa2+ channelGIRKIon Channel Modulation in Pacemaker CellsNorepinephrine AcetylcholineL-type Ca2+ channel conductance  conductanceT-type Ca2+ channelnone noneHyperpolarization-activated channel activation  activationDelayed rectifierK+ channel conductance  conductanceInward rectifierK+ channelnone Opens channelG/GmaxvoltageNAL-typeG/GmaxvoltageNAHCNL-type Ca2+ channels K+ channelsdelayed rectifier Ih activationT-type Ca2+ channels L-type Ca2+ channelsModulation of pacemakerNAL-type Ca2+ channelsK+ channels delayed rectifier inward rectifier Ih activationT-type Ca2+ channels L-type Ca2+ channelsModulation of pacemakerAchDiFrancesco D et al, 1989Speeding and slowing of the pacemakerChannel ModulationRegulation of the M current in neuronsThe discovery of M currentBrown & Adams, 1980Delmas & Brown, 2005Sympathetic, cortical, hippocampal neurons etccontrol M1 muscarinic Ach receptorM current ‘clamps’ the membrane potential-90 mV -45 mVBrown, 1988Delmas & Brown, 2005M1 agonist inhibits M current by decreasing PoBrown, 1988Delmas & Brown, 2005+GqPLCGM1 muscarinic Ach receptor signaling pathwayAchM1 muscarinicAch receptorPIP2Phosphatidylinositol 4,5-bisphosphate1% of membrane phospholipids DAGdiacylglycerolIP3Inositol trisphosphate +PKC+IP3 ReceptorCa2+ release fromintracellular store•Re-synthesis of PIP2 is required for recovery of M current after M1 AchR-mediated inhibition.•M channels require PIP2 to stay open.In inside-out patch, PIP2 analogue prevents M current run-down while PIP2 sequesters accelerate it.•A 3-fold elevation of resting PIP2 level reduces M-current inhibition by M1 AchR. •KCNQ2/3 channels carry neuronal M current.PIP2-binding domain identified.Gating of M channels by PIP2Suh & Hille, 2002Zhang et al., 2003Winks et al., 2005Schenzer et al., 2005Winks et al., 2005M1 AchR activation   membrane PIP2 level  M current+GqPLCGM1 Ach receptor modulation of membrane excitabilityAchM1 muscarinicAch receptordepletion ofPIP2Phosphatidylinositol 4,5-bisphosphate DAGdiacylglycerolIP3Inositol trisphosphate +PKC+IP3 ReceptorCa2+ release fromintracellular storeInhibition of KCNQ/M currentIncrease ofmembrane excitabilityPIP2 modulates multiple channelsSuh & Hille, 2008Spatial and temporal control of channel modulationMultiple channels are modulated in response to one agonist. Individual channel is modulated by multiple signaling pathways.Channel-protein complexes help maintain spatial and temporal specificity of channel modulation.voltage-dependentactivationmillisecondsPKA-dependentmodulationsecondsG-mediated modulation30-100 msG-mediated modulation: a membrane-delimited shortcutPKA-ATPmuscarinicAch receptorAchadenylylcyclaseGiGcAMPCa2+ channelGIRKPKA/CaN modulation of L-type Ca channelsOliveria SF et al, 2007LZ: leucine zipper dimerization domainAKAP: A-kinase anchor proteinCaM: calmodulin (Ca-binding protein)CaN: calcineurin (phosphatase)PKA: protein kinase AL-type Ca channelChannel modulationslow onset and long-lastingmany modulators, many channelsspatial and temporal specificityalteration of membrane excitabilitySpatial Distribution of ChannelsThe spatial distribution of ion channels play important role in controlling neuronal excitability and their computational power.Action potential (AP) generation at axon initial segment (AIS) AP backpropagation from soma to distal dendritesLai H and Jan L,2006Compartmentalization of neuronsand regional distribution of ion channelsPioneering work in spinal motor neurons suggested the AP is generated in the axon initial segment or possibly the first nodes of Ranvier.Coombs et al., 1957Fatt, 1957Fuortes et al.Use of dV/dt to dissociate the individual components that make up the somatic APSimultaneous axonal and somatic recordings in