Pharmacological And Membrane Potential Modulation Of TRPV1Channel, M/Kv7K⁺Channel, Membrane PIP₂and The Physiological Significance

TRPV1is activated by vanilloids (such as capsaicin), low pH (<pH5.8)and noxious heat (>43°C) and a number of endogenous ligands such ascertain inflammatory lipoxygenase products and the endocannibinoidanandamide. TRPV1is considered to be one of the major molecularmechanisms for nociception. Important regions or amino acids in TRPV1possibly involved in capsaicin binding, proton and noxious heat activation,sensitization/desensitization, and phosphorylation of the channel have beendescribed. TRPV1is believed to be activated by stimuli with differentmechanisms. For example, capsaicin was found to bind to the intracellular sitewhereas proton was suggested to bind to the extracellular site of the channel.One striking feature of TRPV1is that the activated currents can be sensitizedand desensitized. This feature implies that TRPV1function is under greatmodulation, which has significant implications for the involvement of TRPV1in physiological and pathophysiological conditions.KCNQ genes encode K⁺channel subunits of the Kv7family. There arefive members of this family: Kv7.1to Kv7.5(KCNQ1-KCNQ5). Of thesemembers, KCNQ1encoded Kv7.1is mainly expressed in cardiac myocytes,and the Kv7.1/KCNE1channel complex that underlies the delayed rectifierpotassium current, IKs, is key for controlling the duration of the actionpotential of the human heart. Kv7.2and Kv7.3are the principal molecularcomponents of the slow voltage-gated M-channel, which widely regulatesneuronal excitability. KCNQ4expression is considered to be restricted to theinner ear and auditory nerves. KCNQ5is broadly expressed in nerves and canalso contribute to M-channels. Recently, KCNQ1, KCNQ4and KCNQ5, butnot KCNQ2and KCNQ3gene expression has been identified in multiple vascular smooth muscle cells. In addition, growing evidence suggested thatKv7channels play a crucial role in regulating vascular contractility.Based upon the above mentioned channels, in my thesis, we usetechniques such as patch clamp, TEVC, and others to systematically study thefollowing subjects:(1) Agonist-dependent potentiation of vanilloid receptorTRPV1function by stilbene derivatives;(2) Modulation of the excitability ofsmall DRG neurons by tannic acid and the ionic mechanisms;(3) Modulationof Kv7K⁺channel by tannins;(4) Mdulation of PtdIns(4,5)P₂level and Kv7currents by membrane depolarization and the mechanisms.Part1Agonist-dependent Potentiation of Vanilloid Receptor TRPV1Function by Stilbene DerivativesObjective: We aim to investigate modulation of TRPV1function bystilbene derivatives DIDS and SITS in adult rat DRG neurons and HEK293expressing system.Methods: Perforated patch clamp was used to monitor the effects ofstilbene derivatives DIDS and SITS on TRPV1channels in DRG neurons orHEK293expressing system. The studies were performaed both in the absenceor presence of extracellular Ca²⁺.Results:(1) DIDS enhanced capsaicin-evoked TRPV1current in adult ratDRG neurons. The effects of DIDS on potentiation of capsaicin-inducedcurrent were concentration dependent, with an EC₅₀of4.66±0.77μM. Onaverage, at saturating concentration DIDS increased capsaicin-inducedcurrents by more than two folds in comparison with the capsaicin-inducedcurrent after taking into account of the current tachyphylaxis. DIDS did notinduce any currents when applied alone. DIDS do not affect thecapsaicin-induced tachyphylaxis. DIDS accelerated the activation of thecapsaicin-induced currents.(2) In Ca²⁺free extracellular solution, DIDSpotentiated the capsaicin-induced currents with an EC50of4.88±0.74μM),similar to the EC₅₀in the presence of extracellular Ca²⁺.(3) DIDS (10μM)strongly enhanced the anandamide-induced TRPV1currents and did not affectthe desensitization.(4) DIDS concentration-dependently increased the low pH-induced current with an EC₅₀of1.83±0.29μM. The low pH-inducedcurrents were increased by448±69%of the control with co-application of100μM DIDS. Furthermore, DIDS almost completely abolished thetachyphylaxis of currents evoked by low pH, in sharp contrast with theno-effects of DIDS on the tachyphylaxis of the capsaicin-induced currents.The activation process of low pH-induced currents was also accelerated byDIDS.(5) DIDS selectively sensitized TRPV1currents but not theacid-sensitive currents induced by low pH.(6) The effects of DIDS are not aresult of its hydrolysis product.(7) SITS (100μM) did not potentiate thecapsaicin-evoked TRPV1currents, but strongly and concentration dependentlypotentiated the low pH-induced currents (8) DIDS enhances currents ofTRPV1expressed in HEK293cells. DIDS potentiated the capsaicin-inducedcurrents in the absence of Ca²⁺with an EC₅₀of3.21±0.10μM.Co-application DIDS with capsaicin led to a leftward shift of theconcentration-response curve for capsaicin activation of TRPV1.100μMDIDS shifted the V_1/2from40±5mV to-42±7mV.(9) DIDS potentiates theTRPV1currents through a mechanism different from the known mechanism.Conclusions:(1) DIDS enhances capsaicin-evoked TRPV1current inadult rat DRG neurons.(2) The effect of DIDS on capsaicin-induced TRPV1currents is similar in the presence or absence of extracellular Ca²⁺.(3) DIDSenhances TRPV1current induced by endovanilloid anandamide.(4) DIDSenhances low pH-evoked TRPV1current and blocks the tachyphylaxis of thelow pH-induced currents.(5) The acid-sensitive currents are not involved inthe DIDS-potentiated currents induced by low pH.(6) The effects of DIDS arenot a result of its hydrolysis product.(7) SITS potentiates the low pH-evokedbut not the capsaicin-evoked currents.(8) DIDS enhances currents of TRPV1expressed in HEK293cells.(9) DIDS does not potentiate TRPV1currentsthrough known modulation sites.Part2Tannic acid inhibits the excitability of small DRG neurons and theunderlying ionic mechanismsObjective: We aim to study the effects of tannic acid on excitability of small DRG neurons and the ionic mechanism.Methods: Perforated patch clamp technique was used to study the effectof tannic acid on excitability of small DRG neurons and the ionic mechanism.Results:(1) In peripheral small DRG neurons, external application of1μM or10μM tannic acid reduced fired frequency and widened the intervals ofof the acion potentials. Significant hyperpolarization from the restingmembrane potentials was observed with applications of retigabine but not oftannic acid.(2) Extracellular application of tannic acid quickly and reversiblypotentiated Kv7/M currents. The action of tannic acid reached its maximumsteady-state value after about2min of drug exposure. The effect of tannic acidon Kv7/M current was concentration dependent, with an EC₅₀value of4.44±0.10μM. On average, at saturating concentration, tannic acid increasedKv7/M currents by about one fold.(3) Tannic acid concentration-dependentlypotentiates Kv7.2and Kv7.2/7.3currents expressed in HEK293cells with anEC₅₀of5.40±0.82μM for Kv7.2and7.38±1.57μM for Kv7.2/7.3. Onaverage, at saturating concentration, tannic acid increased Kv7.2andKv7.2/7.3currents by more than two folds. Tannic acid potentiatedKv7.2(W236L) current; No significant difference for the effects of tannic acidon wildtype Kv7.2current and Kv7.2(W236L) was observed (Kv7.2WT:1.34±0.04fold; Kv7.2(W236L):1.53±0.20fold). Thus, Trp236in KCNQ2is notcritical for tannic acid-induced activation of the channels.(4) Tannic acid (10μM) significantly inhibits bradykinin-enhanced excitability in small DRGneurons. Tannic acid (10μM) significantly inhibits Kv7/M blocker XE991(3μM)-enhanced excitability in small DRG neurons.(5) Tannic acidconcentration-dependently inhibits TTX-sensitive Na⁺currents in small DRGneurons with an EC₅₀value of5.25±0.26μM. At saturating concentration,tannic acid totally blockes TTX-sensitive Na⁺currents.(6) Tannic acidconcentration-dependently inhibits outward K⁺currents in small DRG neuronswith an EC₅₀value of6.55±1.18μM.Conclusions:(1) Tannic acid significantly inhibits spontaneous repetitivefiring of peripheral small DRG neurons.(2) Tannic acid concentration-dependently potentiates Kv7/M-type K^<sup>+currents in small DRGneurons.(3) Tannic acid concentration-dependently potentiates Kv7.2andKv7.2/7.3currents expressed in HEK293cells.(4) Tannic acid significantlyinhibits bradykinin-enhanced excitability in small DRG neurons.(5) Tannicacid concentration-dependently inhibits TTX-sensitive Na⁺currents in smallDRG neurons.(6) Tannic acid concentration-dependently inhibits outward K^<sup>+currents in small DRG neurons.Part3Modulation of Kv7K^<sup>+channels by tanninsObjective: We aim to study the effects of tannins on Kv7K⁺channelcurrents expressed in HEK293cells.Methods: Patch clamp technique was used to study the effect of taninson Kv7K^<sup>+channel currents expressed in HEK293cells.Results:(1) Tannic acid concentration-dependently potentiatedKv7.1/KCNE1K⁺channels expressed in HEK293cells, with an EC₅₀of5.16±0.43μM. On average, at saturating concentration, tannic acid increasedKv7.1/KCNE1K⁺currents by more than two folds. Furthermore, tannic acidslowed the deactivation processes of Kv7.1/KCNE1K⁺currents.(2) Tannicacid slighty inhibited IKs currents in ventricular muscle cells of guinea pig.(3)Tannic acid concentration-dependently inhibited Kv7.1K⁺currents. Tannicacid concentration-dependently potentiated Kv7.3/7.5K⁺channels expressedin HEK293cells.(4) Tannic acid concentration-dependently potentiated Kv7.4K⁺channels expressed in HEK293cells with an EC₅₀of27.3±3.6μM. Onaverage, at saturating concentration, tannic acid increased Kv7.4K⁺currentsby about four folds. The effect of tannic acid on Kv7.4and Kv7.3/7.5K⁺channels may underly the vasodilatory effect of tannins.(5)10μM quercetin,10μM catechin and10μM resveratrol did not affect Kv7.1/KCNE1K⁺current.(6)10μM quercetin,10μM tannic acid and10μM retigabineincreased Kv7.2/7.3K⁺current by128±18%,130±13%,243±31%,respectively.10μM catechin inhibited Kv7.2/7.3K⁺current by40±8%.10μM resveratrol did not affect Kv7.2/7.3K⁺current (16±6%).(7)10μMquercetin,10μM tannic acid and10μM retigabine increased Kv7.4K⁺current by87±10%,146±46%,297±13%, respectively.10μM catechin,10μMresveratrol did not affect Kv7.4K⁺current.Conclusions:(1) Tannic acid can significantly potentiate Kv7.1/KCNE1K⁺channels expressed in HEK293cells, and the effect of tannic acid isconcentration-dependent.(2) Tannic acid inhibits IKs currents in ventricularmuscle cells.(3) Tannic acid inhibits Kv7.1, but potentiates Kv7.3/7.5K⁺channels expressed in HEK293cells.(4) Tannic acidconcentration-dependently potentiates Kv7.4K⁺channels expressed inHEK293cells.(5) Quercetin, catechin and resveratrol did not affectKv7.1/KCNE1K⁺current.(6) Quercetin potentiated Kv7.2/7.3K⁺current.Catechin inhibited Kv7.2/7.3K⁺current. Resveratrol did not affect Kv7.2/7.3K⁺current.(7) Quercetin potentiated Kv7.4K⁺current. Catechin, resveratroldid not affect Kv7.4K⁺current.Part4Depolarization increases PtdIns(4,5)P₂level and Kv7currentsthrough PI4kinase mechanismsObjective: We aim to study modulation of membrane PIP₂by membranedepolarization and the underlying molecular mechanisms.Methods: Two electrode voltage clamp (TEVC) and thin layerchromatography (TLC) techniques were used to investigate modulation ofKv7.2/7.3, Kir2.1, Kir2.3channel current and membrane PIP₂bydepolarization in Xenopus oocytes.Results:(1) Membrane depolarization augments the amplitude ofKv7.2/7.3currents expressed in Xenopus oocytes. The depolarization-inducedpotentiation of Kv7.2/7.3currents was clearly voltage-dependent. The voltagethat produced a half-maximum increase (V_1/2) was–26.1±0.5mV (n=5-14).Depolarization did not affect the kinetics of Kv7.2/7.3currents. The activation(Ï„ act) and deactivation (Ï„ deact) time constants of Kv7.2/7.3currentsmeasured either before or after the currents had been increased by thedepolarization (0mV,15min) were not significantly different (Ï„ act:172±4ms,177±7ms；τ deact:163±3ms,162±10ms). Similarly, theconductance-voltage relationship of Kv7.2/7.3activation was also not affected (control:-28.7±0.5mV,15min depolarization:-29.6±0.4mV).(2)Homomeric Kv7.2currents were also sensitive to depolarization. Actually,Kv7.2currents were increased to a larger extent by depolarization thanKv7.2/7.3currents. However, the voltage-dependence of the induced increaseswas similar for both Kv7.2and Kv7.2/7.3currents (V1/2is-29.1±2.4mV forKv7.2and–26.1±0.5mV for Kv7.2/7.3).(3) Incubation with high K⁺(ND96K) for15min led to potentiation of Kv7.2/7.3currents. The averagefold increase induced by ND96K incubation was similar to that induced by thedepolarization at0mV (183±14%vs.205±6%, for ND96K and0mV,respectively). Similar to voltage-clamp depolarization, high K⁺-induceddepolarization did not affect the activation properties of KCNQ2/Q3currents(control: V1/2=-28.7±0.3mV,15min incubation: V1/2=-27.2±0.5mV). Theabove results suggest that depolarization per se, and not increasing K⁺outflux,contributes to the observed potentiation of Kv7.2/7.3currents.(4) Activationof Ci-VSP interrupted but did not cancel the final potentiation capacity of thedepolarization on Kv7.2/7.3currents. The identity of PIP and PIP₂wasconfirmed with mass spectrometry. High K⁺solution (ND96K) incubationincreased PIP and PIP₂levels by49±4%and43±7%, respectively. Similarly,depolarization at0mV also increased PIP and PIP₂levels by71±9%and55±10%, respectively. The membrane depolarization increased the activity ofboth Kir2.1and Kir2.3channels. Furthermore, the depolarization increasedKir2.3currents more than it did to Kir2.1currents, consistent with the fact thatKir2.3has a lower apparent affinity than Kir2.1with PIP₂. Overall, the aboveresults suggest that the depolarization increases Kv7currents by elevatingPIP₂levels in the oocytes.(5) Incubation with wortmannin, a blocker of PI4kinase greatly reduced the depolarization-induced enhancement of Kv7.2/7.3currents. Injection of the dsRNA for PI4kinase abolished thedepolarization-induced increase in PI4kinase expression. In agreement withthese results, the dsRNA completely abolished the depolarization-inducedpotentiation of Kv7.2/Kv7.3currents. Furthermore, the dsRNA also proventeddepolarization-induced membrane PIP₂increase.(6) Low frequency (10Hz) of "physiological" membrane potential activity (mimicking neuronal actionpotential) already increased Kv7.2/7.3currents compared with quiescent cells(0Hz, held at-70mV). This stimulation at higher frequency (20Hz) furthersignificantly increased Kv7.2/7.3currents.Conclusions:(1) Membrane depolarization augments the amplitude butdoes not affect the kinetics of Kv7.2/7.3currents expressed in Xenopusoocytes.(2) Depolarization induced larger potentiation of homomeric Kv7.2currents.(3) External high potassium depolarizes the membrane potential andincreases Kv7.2/7.3currents.(4) Depolarization increases Kv7.2/7.3currentsthrough increasing membrane PIP₂levels.(5) Depolarization increases PIP₂levels through increased activity of PI4kinase.(6) Physiological stimulationmimicking action potentials frequency-dependently increase Kv7.2/7.3currents.