Study On The Mechanism For Receptor-mediated Modulation Of Kv7/M Potassium Channels And For Regulation Of Volume-activated Chloride Channels

M channels are wildly distributed voltage-dependent potassium channels in neuronal systems that were first described by Brown and Adams in 1980 s. They are characterized by slow-activation,slow-deactivation and non-inactivation. M channels are the only channels activated around threshold potential of neurons. It is now well established that the homomeric and heteromeric Kv7 family channels(Kv7.2,Kv7.3and Kv7.5) underlies the molecular basis of M currents. Functional depression of M channels could increase excitability of the neurons,leading to the diseases such as epilepsy. It is known that many neuronal transmitters and peptides could regulate the M currents thereby affecting the excitability of neurons. Recently, great achievements have been made towards understanding the molecular mechanisms for M current regulation, represented by the mechanisms underling the M current regulation by two G protein coupled receptor pathways, M type acetylcholine receptor(M1) and type II bradykinin receptor(B2).Although activation of both receptors could induce M current inhibition,the underlying molecular mechanisms are different, with key difference in involving intracellular Ca2+ concentration. However, the mechanism which underlies this difference is still not clear. The first part of this thesis will focus on the mechanism for M1- and B2-mediated modulation of Kv7/M channels in rat superior cervical ganglion(SCG) neurons and investigates the roles of lipid rafts in the modulation.Volume-activated chloride channels(VACC) were first found in embryonic rat aortic vascular smooth muscle cells(A10 cells). Further research demosntrate that almost all vertebrate cells produce VACC inresponding to volume expansion. VACC are important for maintaining cell volume and ionic balance, and thus play roles in the cell proliferation and differentiation, apoptosis, vascular myogenic response, hormone secretion and transmembrane chloride ion transport. In addition, VACC are also involved in the anti-cancer drug resistance, excitatory neurotoxicity, ischemic injury and inflammation process.The molecular basis of VACC is not yet conclusive. P-glycoprotein,nucleic acid-sensitive chloride channel protein, voltage-dependent chloride channel Cl C2 and Cl C3, as well as calcium-activated chloride channel(bestrophin and TMEM16A) were all considered to be involved in the consititution of VACC.It is also a debeting issue about the mechanism of VACC activation.Cell swelling is essential for activation of VACC, but it is not clear how exactly the activation of VACC is triggered. Intracellular Ca2+, ATP release, many protein kinases, intracellular ionic strength and lipid rafts have ever been indicated in the activation of VACC.The second part of the thesis will study the VACC in rat dorsal root ganglion(DRG) neurons and will focus on the pharmacological characteristics and molecular basis of the VACC. The third part will study the activation machanism of VACC in DRG neurons and will focus on the role of intracellular Ca2+ and PLC in the activation of VACC.Part 1 Mechanism for specificity of receptor-mediated modulation ofKv7/M potassium currents in rat SCG neuronsObjective: We aim to investigate the role of membrane microdomain in the specificity of receptor-mediated modulation of Kv7/M potassium currents in rat SCG neuronsMethods: Perforated patch clamp was used to record Kv7/M current in rat SCG neurons;(2) Membrane lipid raft was isolated using sucrose density gradient centrifugation to observe the distribution of receptors and related signaling proteins;(3) Intracellular Ca2+ was imaged using confocal microscopy;(4) Immunoprecipitation was used to detect theinteraction between IP3 R and B2 R.Results:(1) Bath application of Oxo-M(M cholinergic receptor agonists) or BK(bradykinin, B2 receptor agonists) caused a concentration-dependent inhibition of Kv7/M currents, with a 50%effective concentration(EC50) of 0.44 ± 0.09 μM and 1.73 ± 0.77 n M for Oxo-M and BK, respectively. The maximal inhibition of Kv7/M currents by 5 μM Oxo-M and 100 n M BK amounted to 82 ± 6.3% and 77 ± 7.1%,respectively. MβCD could extract the membrane cholesterol thus disrupting the lipid raft. When the neurons were treated with 5 m M MβCD first, the Oxo-M-induced inhibition of Kv7/M current was not affected, whereas the BK-induced inhibition was significantly reduced.When neurons were treated with 10 m M MβCD first, both Oxo-M and BK-induced inhibitions of Kv7/M currents were reduced. An inactive analog of MβCD, αCD, did not affect Oxo-M or BK-induced inhibition of the Kv7/M current;(2) After the nine sequential fractions were obtained from the sucrose gradient experiments, caveolin-1, which is the marker of lipid raft, was found distributed in low buoyancy density fractions(fraction 3-5). Immunoblotting results also showed the presence of B2 R,Gq, G11, PLCβ1 and PLCβ4 in the lipid raft-containing fractions. However,M1 R was not detected in the lipid raft fractions. When the SCG tissue was pretreated with 5 m M MβCD, lipid raft was disrupted that lead to the move of caveolin-1 to high density fractions accompanied with B2 R.Conversely, BK treatment increased the B2 R distribution in the lipid raft fractions. In contrast, M1 R activation did not alter the distribution of M1 R in the raft fractions;(3) BK induced an increase of intracellular Ca2+,which was significantly reduced by treatment with 5 m M MβCD. In the absence of extracellular Ca2+, BK still induced an rising of intracellular Ca2+ which was abolished by pretreatment with MβCD; when the Ca2+store was depleted with thapsigargin first, BK again induced an rising of intracellular Ca2+, which however was potentiated rather than inhibited by MβCD treatment;(4) B2 R, but not M1 R, was detected in IP3 Rimmunoprecipitates, and B2 R was not co-immunoprecipitated with IP3 R after treatment with MβCD.Conclusion:(1) Lipid rafts play key roles in B2R-mediated modulation of Kv7/M channels in SCG neurons;(2) Lipid raft microdomain is present in rat SCG tissue cells. B2 receptor and associated signaling molecules such as Gq, G11, PLCβ4 and PLCβ1 are found in the lipid raft fractions; M1 receptor is not found in rafts;(3) B2R-induced intracellular Ca2+ rise is dependent on the integrity of lipid raft microdomain, whether the Ca2+ is from the internal store or the influx from a external source;(4) The interaction between IP3 R and B2 R depends on the integrity of lipid raft microdomain.Part 2 The characteristics and molecular basis of volume-activatedchloride current(VACC) in DRG neuronsObjective: We aim to characterize the VACC in rat DRG neurons and study its molecular basis.Methods:(1) A hypertonic piette solution(420 m Osm containing Cs Cl) and an isotonic bath solution(320 m Osm containing Na Cl) were used to record the VACC from rat DRG neurons and CHO cells using whole cell patch clamp technique;(2) si RNA were constructed in lentivirus and used to depress TMEM16 A or Bestrophin-1 in DRG neurons. Q-PCR was used to assess the efficiency of the si RNAs.Results:(1) VACC was elicited as an inward current at-60 m V using the internal and external solutions described in the method, and also an increase of cell volume was seen accomping the developnet of the VACC. VACC was present in all types of DRG neurons and the avarage current density was about 11 p A/p F. The VACC showed the characteristic of outward rectification when the currents were induced by a-100 m V to+100 m V ramp voltage prtocol. In addition, the currents also included rapid-activation and rapid-deactivation properties when the currents were induced by a step depolarization voltage protocol. The currents began to show inactivation when induced at potentials more positive than 60 m V.VACC was abolsihed when the hypertonic internal Cs Cl was replaced by Cs2SO4, or when isotonic internal solution was used. VACC could be blocked by NPPB, a chloride channel blocker, and the current blocked by NPPB reversd at about 0 m V which was close to the chloride ion equilibrium potential(-2 m V).(2) VACC was sensitive to the commonly used chloride channel blockers, including NFA, NPPB, tannic acid, DIDS and CACC-A01. The inhibition of VACC by these blockers at concentrtion of 100 μM was 51.7 ± 7.4%, 79.9 ± 4.7%, 75.5 ± 2.8%, 18.1± 4.5%, 87.4 ± 4.7%, respectively; CACC-A01 was most and DIDS was least effective in blocking VACC.(3) Including 20 m M BAPTA but not EGTA in the hypertonic pippete solution abolished the VACC; the current density was reduced significantly when the DRG neurons were pretreated with thapsigargin.(4) VACC was reduced significantly when the neurons were transfected first with TMEM16 A si RNA. However,Bestrophin-1 si RNA did not affaect VACC current density.(5)Bestrophin-1 could be activated by Ca2+ influx through the voltage-gated Ca2+ channel(VDCC), and this Bestrophin-1 current was different from the VACC.(6) VACC could be induced by internal hypertonic pipette solution from CHO cells when overexpressed with TMEM16 A, and the properties of this VACC were similar to that of DRG neurons. Similar VACC in CHO was abolished by internal BAPTA, but not by EGTA.Conclusion:(1) VACC is induced in rat DRG neurons by a hypertonic internal solution containing Cs Cl. The VACC is sensitive to the commonly used chloride channel blockers, but is less sensitive to DIDS.(2) A locolized rising of intracellular Ca2+ is essential for the activation of VACC in DRG neurons.(4) TMEM16 A, but not bestrophin-1 is possibly one of the molecular component of VACC in rat DRG neurons.Part 3 Activation mechanism of volume-activated chloride currentsin rat DRG neuronsObjective: We aim to investigate the activation machanism ofVACC in rat DRG neurons.Methods:(1) Patch clamp technology was used to record the VACC from DRG neurons and from HEK293 cells;(2) si RNA were constructed in lentivirus and used to depress PLCβ3 in DRG neurons. Q-PCR was used to assess the efficiency of the si RNAs.Results:(1) Reduce extracellular Ca2+(chelated with 2 m M EGTA)in the bath solution did not affect the VACC induced by hypertonic internal solution; internal perfusion of DRG neurons with a isotonic pipette solution containing high Ca2+(7.4 μM) did not induce VACC.(2)20 U/ml apyrase, a enzyme hydrolyzing ATP, had no effect on VACC, and ATP-free pipette solution still induced VACC; direct application of 100μM ATP could not activate the VACC; purinergic receptor blocker suramin(100 μM) inhibited the VACC voltage-dependently, with a 27.65± 4.36% inhibition at-60 m V and 77.38 ± 8.83% inhibition at +100 m V,respectively.(3) The VACC in CHO-TMEM16 A cells was also inhibited by suramin similarly as that in DRG neurons; more importantly, when the TMEM16 A current was activated not by hypertonic solution but by a high internal free Ca2+(447 n M), suramin also block the TMEM16 A currents with similar voltage-dependency.(4) Including 500 μM GDP-βs in the pipette solution did not affect the activation of VACC.(5) After the lipid raft was disrupted by 5 m M MβCD, the VACC current density was increased in DRG neurons.(6) Pretreatment of the DRG with PLC inhibitor edelfosine or U73122 reduced the VACC significantly, but the inactive analog of U73122, U73343, did not affect the current.(7) VACC could also be elicited by a hypotonic bath solution(HTS solution), and this VACC was also inhibited by BAPTA; the HTS-induced intracelluar Ca2+ rise could be blocked by U73122.(8) The VACC in DRG neurons was reduced largely when the neurons were treated with PLCβ3 si RNA.(9) Large VACC could also be elicited in HEK293 A cells which was abolished by 20 m M BAPTA in pippete solution. ATP-free pipette solution did not prevent the development of VACC. Direct application of100 μM ATP did not induce VACC-like currents in HEK293 A cells under isotonic conditions. Suramin inhibited VACC in HEK293 A voltage-dependently.Conclusion:(1) The activation of VACC in DRG neurons depends on a localized but not a gross increase of intracellular Ca2+ level;(2) ATP and G poteins are not involved in the activation of VACC in rat DRG neurons. Suramin blocks the VACC(TMEM16A) directly;(3) Disruption of lipid raft significantly increases the VACC;(4) Activation of PLC,more specifically PLCβ3 may participate in the activation of VACC;(5)HEK293A has largly endogenous VACC, the properties and activation mechanism of which are similar to that in DRG neurons.