Receptor-Mediated And Pharmacological Modulation Of M/KCNQ K⁺ Channel, Ca²⁺ Activated Cl^- Channel, Kir Channel And The Physiological Significance

M type potassium channel conducts a voltage and time dependent, slowly activating, slowly deactivating and non-inactivating outward current. It is such named since it can be robustly inhibited by muscarinic M1 receptor activation. At present, it is generally accepted that the molecular basis of M channel is constituted by homo- and hetero-multimers of KCNQ (KCNQ1-5) subunits. M channel opens at a threshold below -60 mV and this feature allows for a fraction of these channels to be open at or near the resting membrane potential of a neuron. It is suggested that M current in neurons can serve as an intrinsic"voltage-clamp"mechanism controlling the resting membrane potential and the threshold for action potential firing thus repressing the over excitability of neurons. M channel is extensively distributed in nervous system, including hippocampus, cortical, sensory and some sympathetic neurons. Moreover, growing evidence suggests that functional M channels are expressed in the peripheral sensory fibers and their activity strongly contributes to the fiber excitability in vivo.Ca2+ activated Cl- channels (CACC) are a prominent group of ion channels robustly expressed in many mammalian cell types including neurons, smooth muscles and epithelia. In the nervous system, functional CaCCs are best characterized in neurons mediating different sensory inputs, such as olfactory neurons, photosensitive rods and cons, taste calls and DRG neurons. CaCCs play a role in depolarization or amplification of a depolarizing input produced by other channels in the nervous system. The molecular identity of CaCC remained controversial until very recently when three groups independently identified a member of the transmembrane protein 16A (TMEM16A) protein family as a CaCC subunit. This important finding makes it possible to alter CaCCs in native cells via genetic manipulationsInwardly rectifying potassium (Kir) channel family contains at least 7 subfamilies (Kir1-7). These channels are extensively distributed in various tissues and serve a role in maintaining K+ equilibrium and keeping membrane potential. These channels play an important role in the repolarization phase of action potential and regulation of cell excitability. Furthermore, recent studies indicate PIP2 may play an important role in modulation of Kir2.0 channel function by several modulators.Based upon the above mentioned channels, in my thesis, we use techniques such as patch clamp, calcium imaging, siRNA, native neuron transfection, TEVC, LSCM and animal behavioral assay, etc. to systematically study the following subjects: (1) Ionic mechanisms for the acute nociceptive signals induced by inflammatory mediator bradykinin (BK); (2) Mechanism underlying inflammatory mediator histamine regulation of KCNQ/M channels; (3) Mechanism underlying the first-generation antihistamine induced severe neuronal adverse effects.Part 1 Ionic mechanisms for the acute nociceptive signals induced by bradykininObjective: We aim to investigate the mechanisms of acute nociceptive signaling induced by bradykinin. We characterized major membrane currents directly affected by BK in nociceptive sensory neurons, investigated their molecular identities, signaling cascades coupling their regulation to BK receptors and the contribution of these currents to the excitability of nociceptive neurons and to the BK-induced nocifensive behaviour in rats.Methods: Perforated patch clamp (voltage clamp) was used to monitor the effects of BK on ion channels in DRG neurons. Current clamp was used to record the neuron excitability. Calcium photometry was used to measure the changes of intracellular Ca2+ concentration. Membrane PIP2 hydrolysis was monitored by confocal microscope. Whole animal behavioral study was carried out to monitor the nocifensive behavior of rats upon BK application.Results: (1) BK inhibits M current in small DRG neurons: BK caused a prominent concentration-dependent inhibition of M current in small DRG neurons, with an EC50 of 60.0±16.3 nM. The inhibitory effect was independent of non-neuronal cells since purified neuron culture did not affect BK's response. (2) Signaling pathway of BK-induced M current inhibition: BK produced significant phospholipase C (PLC) activation since PLCδ-PH-GFP, a fluorescent probe that binds PIP2 and IP3, translocated from the membrane to the cytosol after BK application. However, membrane PIP2 concentration did not drop significantly since a more specific PIP2 probe YFP-tubby failed to translocate to the cytosol following BK application. Ca2+ imaging experiments showed that BK induced significant intracellular Ca2+ rises in DRG neurons. Patch clamp studies further revealed that the inhibitory effect of BK was significantly attenuated by B2R antagonist Hoe-140, PLC inhibitor edelfosine, IP3 receptor antagonist Xe-C, SERCA inhibitor thapsigargin and high BAPTA internal solution, but was not affected by PKC inhibitor bisindolylmaleimide and PLA2 inhibitor quinacrine. (3) BK activates TMEM16A-dependent Cl- channels: BK can induce a prominent inward current accompanied by M current inhibition. This inward current was not due to TRPV1 or HCN channel opening since ruthenium red and ZD7288 did not affect it. However, this inward current was abolished by Cl- channel blocker DIDS and low Cl- internal solution. Further experiments showed that the inward current was also abolished by B2R antagonist Hoe-140, PLC inhibitor edelfosine, IP3 receptor antagonist Xe-C, SERCA inhibitor thapsigargin and high BAPTA internal solution. The above results suggest that the inward current was conducted by Ca2+ activated Cl- channels (CACC). The gene encoding essential CaCC subunit has recently been identified as Tmem 16a. Experiments using siRNA against Tmem 16a significantly attenuated BK induced inward current, revealing that Tmem 16a is a molecular correlate of the BK-induced Cl- current in DRG neurons. (4) M-current inhibition and CaCC activation contribute to and can account for the excitatory effect of BK: BK and XE991 could both significantly increase DRG neuron excitability and cause depolarization. In low Cl- intracellular solution which abolished BK-induced inward current, co-application of BK + XE991 failed to produce further excitation upon XE991 application. However, XE991 could further excite the neuron in the presence of BK. (5) M channel openers can avert nocifensive behaviour induced by BK: Intraplantar injection of BK (10 nM/site) into the rat hind paw produced strong nocifensive behaviour, which was significantly alleviated by specific M channel openers retigabine or zinc pyrithione. However, the Cl- channel blocker DIDS had no clear effect on the BK-induced nocifensive behaviour and co-application of retigabine and DIDS had only a marginal additivity.Conclusions: (1) BK concentration-dependently inhibits M current in small DRG neurons. The action of BK on M current is not mediated by non-neuronal cells but due to a primary effect on DRG neurons. (2) BK activates PLC but does not produce a global depletion of the membrane PIP2. BK can also induce intracellular Ca2+ mobilization. Pharmacological studies reveal that the inhibitory effect of BK on M current is due to B2R-PLC and IP3-mediated intracellular Ca2+ rise. (3) A series of pharmacological experiments show that the inward current induced by BK is conducted by CACC. Furthermore, siRNA experiments reveal that the recently discovered Tmem 16a underlies molecular correlate of CACC in DRG neurons. (4) Current clamp studies show that BK causes hyperexcitability in DRG neurons. By comparing the effects of BK and Xe991 in high and low Cl- containing internal solution, we show that M current and CACC are both involved in BK's excitatory effect. (5) BK induced nocifensive behavior in rat is significantly attenuated by M current enhancers, suggesting inhibition of M channels in the peripheral sensory terminals can indeed produce firing of action potentials in vivo.Part 2 Phosphatidylinositol 4, 5-bisphosphate hydrolysis mediates histamine-induced KCNQ/M current inhibitionObjective: We aim to study the effects of histamine on KCNQ/M channel current in HEK293 cells and rat SCG neurons. We further explore the signaling pathway and compare the effects of histamine on SCG neurons with those of the well characterized M1 and B2 receptor agonists, oxo-M and BK. The final goal will be to understand the common mechanisms underlying GPCR modulation of KCNQ/M channels in more detail.Methods: Patch clamp technique was used to study the effect of histamine on channel currents and calcium photometry was used to measure the changes of intracellular Ca2+ concentration. Pharmacology methods, in combination with LSCM, were used to study the signaling pathway underlying histamine modulation of KCNQ/M channel in detail.Results: (1) Histamine suppresses KCNQ/M current in HEK293 cells and rat SCG neurons via activation of H1 receptor: Histamine (10μM) strongly inhibited KCNQ2/Q3 channel currents heterologusly expressed in HEK293 cells and the inhibition was 77.9±3.5%. The inhibitory effect was not affected by H2 receptor antagonist cimetidine (p > 0.05 vs. control) but was attenuated by H1 receptor antagonist mepyramine (p < 0.01 vs. control). Histamine could also inhibit M channel current in rat SCG neurons in a concentration dependent manner and this effect was also attenuated by mepyramine (p < 0.01 vs. control). The EC50 deduced from the concentration response relationship was 3.3±1.1μM. Moreover, the existence of H1 receptor in SCG neurons was confirmed by Western blot. (2) Histamine suppresses KCNQ/M current via activation of PLC: The inhibitory effect of histamine was significantly reduced to 7.1±0.6% (p < 0.01 vs. control) by a PLC antagonist U-73122. Meanwhile, the inhibitory effect of histamine on M current in SCG neurons was also significantly abolished by U-73122 (p < 0.01 vs. control). (3) Histamine-induced inhibition of KCNQ2/Q3 currents is the result of membrane PIP2 hydrolysis: Histamine produced an obvious translocation of the PLCδ1-PH-GFP probe from the membrane to the cytosol and the effect was reversible upon histamine washout. The rise of fluorescence signals in the cytosol evoked by application of histamine was 80.4±6.0%. This effect was blocked by mepyramine and U-73122 (p < 0.01 vs. control). Patch clamp studies showed that the recovery of the current inhibited by histamine was significantly reduced to 10.5±3.4% (p < 0.01 vs. control) when treated with PI4-Kinase inhibitor wortmannin, compared with 88.4±6.2% in control conditions. In addition, PAO, another PI4-Kinase inhibitor, also significantly reduced the recovery to 6.3±0.9% (p < 0.01 vs. control) compared with 95.4±4.6% in control conditions. (4) Histamine inhibits KCNQ2/Q3 currents in HEK293 cells deprived of [Ca2+]i rises: Histamine (10μM) could induce an obvious [Ca2+]i rise and this effect still existed in Ca2+-free extracellular solution but largely abolished by U-73122. In whole-cell patch clamp configuration, the histamine induced KCNQ current inhibition in HEK293 cells was not altered (81.3±4.0%, p > 0.05 vs. control) when cells were dialyzed with high BAPTA intracellular solution. Besides, thapsigargin, a sarcoplasmic-endoplasmic reticulum Ca2+ pump inhibitor, which can empty the Ca2+ stores, did not affect the effect of histamine, either (88.6±3.9%, p > 0.05 vs. control). (5) H1 receptor stimulation does not evoke [Ca2+]i signal in SCG neurons: Calcium photometry was used to monitor the intracellular Ca2+ changes upon histamine application and the effect of histamine was compared with M1 and BK2 receptor agonist oxo-M and BK. BK could evoke an obvious [Ca2+]i rise, while oxo-M and histamine could not. The rises in the F340/380 ratio produced by histamine, oxo-M and BK were 0.04±0.008, 0.03±0.004 and 0.32±0.05. (6) Histamine does not require [Ca2+]i signal for M current inhibition in SCG neurons: Application of histamine, oxo-M and BK suppressed M currents by 38.3±7.9%, 98.8±0.4% and 85.3±4.4%, respectively, when neurons were dialyzed with low BAPTA internal solution (containing 0.1 mM BAPTA). When neurons were dialyzed with high BAPTA internal solutions, the suppression of M currents by histamine and oxo-M was 32.1±3.1% and 87.3±7.0%, respectively, which was not significantly altered (p > 0.05 vs. control). In contrast, the inhibitory effect of BK was significantly reduced to 20.5±2.6% (p < 0.01 vs. control).Conclusions: (1) Histamine inhibits KCNQ2/Q3 and M channel currents in heterologusly expressed HEK293 cells and rat SCG neurons by H1 receptor activation and this inhibitory effect shows dose dependence. (2) The inhibitory effect of histamine is closely related to PLC activation. (3) Histamine can induce membrane PIP2 hydrolysis and its inhibitory effect on KCNQ currents may likely be due to the hydrolysis of membrane PIP2. (4) The inhibitory effect of histamine on KCNQ currents in HEK293 cells does not rely on intracellular Ca2+ signals despite the fact that it can mobilize intracellular Ca2+ in these cells. (5) The inhibitory effect of histamine on M currents in SCG neurons is also independent of intracellular Ca2+ signals. This effect is similar with that of the oxo-M. Thus activation of H1 receptor inhibits M currents in rat SCG neurons through a similar mechanism with that of M1 receptor. This finding further enriches our understanding of signaling pathway underlying GPCR modulation of KCNQ/M channel. Moreover, the inhibitory effect of histamine on M current may provide new insights into the understanding of the excitatory effects of histamine on neurons.Part 3 Antihistamine mepyramine directly inhibits KCNQ/M channel and depolarizes rat superior cervical ganglion neurons Objective: We aim to study the effects of the first-generationantihistamine mepyramine and diphenhydramine on KCNQ/M channel currents and the underlying mechanisms. We also aim to study their effects on neuron excitability. We try to unravel the molecular mechanism underlying the neuronal toxicity induced by overdose of first-generation antihistamines.Methods: Patch clamp technique was used to study the effect of mepyramine and diphenhydramine on KCNQ/M channel currents and the changes in channel kinetics were analyzed. Whole-cell patch clamp for intracellular drug application and outside-out patch were used to determine the action side of the drug. Current clamp recording mode was used to monitor the effects of drugs on membrane potentials and neuron excitability.Results: (1) Mepyramine inhibits KCNQ2/Q3 channel currents and affects channel gating properties: KCNQ2/Q3 channels were expressed heterologusly in HEK293 cells by means of transfection. The first-generation antihistamine mepyramine could strongly inhibit KCNQ2/Q3 currents and the inhibition rate reached 92.0±1.7%. The inhibition developed rapidly and could be largely washed out. This effect showed concentration dependency and IC50 was 12.5±1.8μM. However, the H2 receptor antagonist cimetidine did not affect KCNQ2/Q3 currents. (2) The effect of mepyramine on channel kinetics: Mepyramine could prolong activation and shorten deactivation kinetics of KCNQ2/Q3 channels and right shift the half-maximal activation (V50) from -16.9±0.9 mV to -7.8±1.4 mV. (3) Diphenhydramine shows similar inhibitory effect. The inhibition produced by 100μM diphenhydramine was 66.9±5.9% and the I-V curve was shifted to the right. (4) Effect of mepyramine on KCNQ families (KCNQ1-5): Mepyramine could inhibit homomeric KCNQ1, KCNQ2, KCNQ3, KCNQ4 and heteromeric KCNQ3/5 channel currents and the inhibitory effects showed concentration dependence. (5) Mepyramine directly inhibits KCNQ2/Q3 channel current from outside of the cell membrane: We included mepyramine in the recording pipette for whole-cell recording to see if direct introduction of mepyramine into the cell would result in KCNQ2/Q3 current inhibition in HEK293 cells. In control and mepyramine treated groups, KCNQ2/Q3 current was decreased to 56.0±9.0% and 54.8±11.6% of its initial level after 12 min dialysis. There was no significant difference between these two groups (p > 0.05). After rundown of KCNQ2/Q3 currents, the inhibitory effect of oxo-M, an M1 receptor agonist that hydrolyzes PIP2 on KCNQ2/Q3 current, was significantly reduced, while effect of mepyramine was not. Outside-out patch clamp studies revealed that mepyramine could quickly and significantly inhibit KCNQ currents (90.1±2.8%) in the membrane excised from HEK293 cells. (6) Mepyramine inhibits M current in rat SCG neurons: Meyramine also inhibited M current in SCG neurons, the inhibition was 76.7±2.6% and showed concentration dependence (IC50 = 25.7±0.7μM). In addition, mepyramine also significantly depolarized the SCG neurons from–51.0±5.6 mV to–38.9±7.3 mV. Mepyramine could not induce depolarization in the continued presence of a specific M channel blocker, linopirdine. (7) Effect of mepyramine on evoked neuronal action potentials: We found that linopirdine could induce remarkable hyperexcitability in SCG neurons. On the contrary, mepyramine did not show any effect on the excitability of neurons.Conclusions: (1) The first-generation antihistamine mepyramine and diphenhydramine can significantly inhibit KCNQ/M currents and this effect is due to direct drug inhibition from the extracellular side. (2) Mepyramine alters KCNQ channel activation and deactivation kinetics and shifts the I-V curve to the right. (3) Mepyramine concentration-dependently inhibits all members of KCNQ family (KCNQ1-5). (4) Mepyramine also inhibits M current in rat SCG neurons and induces depolarization in neurons. However, mepyramine does not evoke hyperexcitability in neurons. These actions are likely to be involved in the adverse neuroexcitatory effects including seizers, convulsion and epilepsy, observed in patients intoxicated by overdose of first-generation antihistamines.Part 4 Selective inhibition of Kir currents by antihistaminesObjective: We aim to study the effects of the first, second and the third-generation antihistamines on inwardly rectifying potassium (Kir) channels and try to reveal the molecular mechanisms underlying neurotoxic effects by overdose of H1 receptor antagonists.Methods: DNA preparation, RNA in vitro transcription and microinjection methods were used to express Kir2.1, 2.3 and 3.4 channels in Xenopus oocyte. Two electrode voltage clamp (TEVC) was used to record the whole cell currents in oocyte and to observe the effects of each type of antihistamines on channel currents.Results: (1) Effect of antihistamines on Kir2.3 currents expressed in Xenopus oocyte: Kir2.3 channel showed a substantial inwardly rectifying property in a high extra-cellular K+ bath solution. The reverse potential was near 0 mV. Extra-cellular perfusion with 100μM mepyramine or diphenhydramine, two histamine H1 receptor antagonist of the first-generation antihistamine, both reduced the current amplitude by 25.0±2.9% and 17.3±0.7%, respectively. However, the second and third-generation H1 receptor antihistamines astemizole, desloratadine and H2 receptor antagonist cimetidine had no effect on Kir2.3 channel current. When deduced from the I-V curve, mepyramine-induced inhibition of Kir2.3 currents was not voltage-dependent. The Kir2.3 current inhibitions induced by mepyramine and diphenhydramine showed concentration dependence and IC50 was 306.4μM and 689.2μM, respectively. (2) Effect of antihistamines on Kir2.1 currents expressed in Xenopus oocyte: Mepyramine and diphenhydramine, at concentrations of 100μM, had little or no inhibitory effect on Kir2.1 channel currents. Similar results were also obtained from other drugs. Astemizole, desloratadine and cimetidine had no effect on Kir2.1 currents. (3) Selective inhibition of antihistamines on Kir currents depends on characteristic channel-PIP2 interaction: We found that the effect of mepyramine could be significantly abolished by a point mutation of Kir2.3, Kir2.3 (I213L), which could confer a stronger channel-PIP2 interaction to Kir2.3 channel. In addition, mepyramine could also inhibit Kir3.4, another member of the Kir subfamily, which has also been shown to be a Kir channel interacting weakly with PIP2 as Kir2.3. Our results indicated the rank order for current inhibition among Kir channels as follows: Kir3.4 > Kir2.3 > Kir2.3 (I213L) > Kir2.1. This result coincided with our previous results showing the rank order of the strength of channel-PIP2 interactions among Kir channels. (4) Inhibition of Kir2.3 current by the first-generation antihistamines leads to the depolarization of resting membrane potential of oocytes: After expressing Kir2.3 channels, the oocytes displayed resting membrane potential (Vm) values of -95.2±2.3 mV. Application of mepyramine (300μM) gradually and significantly depolarized Vm to -57.1±6.1 mV. Similar result was also obtained for 300μM diphenhydramine, which reduced the resting Vm value to -70.0±3.4mV.Conclusions: (1) The first-generation antihistamines mepyramine and diphenhydramine significantly and concentration dependently inhibit Kir2.3 channel current. However, the second and third-generation H1 receptor antihistamine and H2 receptor antagonist astemizole, desloratadine and cimetidine have no effect on Kir2.3 currnet. (2) Kir2.1 channel is insensitive to any antihistamines. (3) The obvious discrepancy of the effect of mepyramine between Kir2.1 and Kir2.3 is due to channel-PIP2 interactions. Mepyramine also inhibits Kir3.4 channel, which shows weak channel-PIP2 interaction. (4) Mepyramine induces a significant depolarization in oocytes by inhibiting Kir2.3 channel. Thus, Kir2.3 channel inhibition may likely be involved in the molecular mechanisms underlying overdose first-generation antihistamine induced neurotoxic effects.SUMMARY1 BK inhibits M-type K+ channel and activates Ca2+-activated Cl- channel in small DRG neurons. BK, acting through its B2 receptors, PLC and releasing of Ca2+ ions from intracellular stores, robustly inhibits M-type K+ channels and activates TMEM 16A-dependent Ca2+-activated Cl- channels. Summation of these two effects adequately accounts for the depolarization and increase in action potential firing induced by BK in DRG neurons. Preclusion of BK-induced inhibition of M channels with specific openers strongly attenuates the nociceptive effect of BK in vivo. The above results reveal the detailed molecular mechanism underlying BK-induced inflammatory pain and provide new directions for the treatment of inflammatory pain.2 Histamine, via activating H1 receptor, PLC and hydrolyzing PIP2, inhibits KCNQ/M channel in HEK293 cells and rat SCG neurons. This process does not involve Ca2+ signal. By comparing the effects of histamine on SCG neurons with those of the well-characterized M1 and B2 receptor agonist, oxo-M, and BK, we suggest that histamine inhibits M current in a similar mechanism with oxo-M.3 The first-generation antihistamine mepyramne and diphenhydramine concentration dependently inhibit KCNQ2/Q3 channel exogenously expressed in HEK293 cells. Meyramine alters channel activation and deactivation kinetics and shift the I-V curve to the right. In addition, mepyramine concentration dependently inhibits all KCNQ channels, including KCNQ1-4 and KCNQ3/Q5. Moreover, mepyrmaine, by inhibiting M channel current in rat SCG neurons, induces significant membrane potential depolarization in neurons. The IC50 for the inhibitory effect of mepyramine lies in the range of plasma drug concentration of intoxicated patients. Thus, KCNQ/M channel inhibition and the subsequent marked depolarization of neuron membrane potential may participate in adverse effects observed in patients intoxicated by overdose with first-generation antihistamines.4 The first-generation antihistamine mepyramne and diphenhydramine dose dependently inhibit Kir2.3 channel current expressed in Xenopus oocytes and induce membrane depolarization, while the second and third-generation antihistamine astemizole and desloratadine do not. Kir2.1 current is insensitive to any of these drugs. Mepyrmaine also inhibits Kir3.4 current. We speculate that the specific Kir channel inhibition produced by the first-generation antihistamine is likely to be determined by the strength of channel-PIP2 interactions.