Study Of Functional Regulation Of KCNQ Channels

The KCNQ genes encoded channels is an important group of potassium channels, which contains five members such as KCNQ1⁵ potassium channel to date. Mutation of KCNQ channels has been implicated in a range of inherited disease. KCNQ1, previously named KVLQT1, was first identified in heart and is responsible for the inherited cardiac arrhythmia called as LQT1 disease. KCNQ2⁵ are mainly expressed in CNS, the inner ear (KCNQ4), and skeletal muscle (KCNQ5). An inherited deafness (DFNA2) is likely to have the relationships with the modulation of the KCNQ4 potassium channel. Heteromeric KCNQ2 and KCNQ3 channels have been proved to underlie the neuronal M-current , which is an important determinant of membrane excitability in many neuronal cells. The mutation of KCNQ2 and KCNQ3 potassium channel leads to a disease, benign familial neonatal convulsions (BFNC). M-current composed of KCNQ2/3 is a voltage-and time-dependent, low-threshold, non-inactivating current. It is a primary potassium current activated at voltage near the threshold for action potential initiation, implying its important role in controlling generation of spikes and neuronal excitability. Since the M-current possesses the unique characteristic of sustained activation and absence of inactivation, this potassium channel is slowly activated when the neuron is depolarized toward the threshold for action potential firing, hyperpolarizing the membrane back toward rest and reducing membrane excitability. So increase of the M-current function would decrease the excitability of neural cells. On the other hands, suppression of the M-current would induce the membrane potential depolarization and induce more action potentials firing, Retigabine activates M-currents and is the first known M-channel opener that has potential applications as anticonvulsant and antiepilepsy agent. Linopirdine and XE991 are M-channel blockers and can increase the CNS excitability, and is suggested to be able to improve the Alzheimer's patient memory and perceptibility. The study of functional modulation of M-channel and KCNQ2/3 is a key step in order to understand the role of M-channel in neuronal physiology and pathophysiology. Although both KCNQ and KV channel families posses a similar topological structure such as 6-TMDS, selectively pore loops and S4 voltage sensor, KCNQ channels show some marked differences from KV channels. First, KCNQ channels lack the conserved N-terminal T1-domain of KV channels; second , KCNQ channels lack of the rapid N-type inactivation mechanism; third, KCNQ channels possess relatively long intracellular C-terminus which often carries motifs for channel modulation. Apart from the structural differences, there are also differences in terms of electrophysiology and pharmacology between KCNQ and KV channels. All these similarities and differences are helpful for our understanding of M-currents. KCNQ potassium channels are readily modulated by extracellular pH, Ca2+, K+ and some other factors. KCNQ channels can also be regulated by a number of neurotransmitters and hormones such as acetylcholine, bradykinin, histamine, angiotensinâ…¡, LHRH (luteinizing hormone-releasing hormone). All of these neuroactive substances can inhibit the M-currents through their Gq coupled receptors. M-current can be enhanced by dopamine D2 receptor activation, which coupled with Gi protein; M-current can also be modulated by channel phosphorylation and dephosphorylation. Because the M-current plays a crucial role in determining neuron excitability, and because understanding its regulation mechanisms has been a long struggle, M-current has become one of the foci of study in neurosscience nowadays. In the present study, KCNQ 2/3 potassium channels were expressed in Xenopus Oocytes, HEK293 and CHO cell lines with methods of RNA injection or DNA transfections. We studied the KCNQ2/3 channel from three aspects. First we studied of the effects of extracelluar pH on KCNQ2/3 currents; second we studied the effects of EGFR, M1, B2 receptors activation KCNQ2/3 currents; Finally we studied of the effects of phosphorylation and dephosphorylation on KCNQ2/3 currents. We concludethat PIP2 and phosphorylation are important factors in the functional regulation of M-channel. 1 The characteristics of KCNQ2/3 potassium channel Aim: To express KCNQ2 and KCNQ3 potassium channels in Xenopus Oocytes and to observe the characteristics of KCNQ2/3 currents by two-microelectrodes voltage clamp (TEVC) method. Methods: All the KCNQ channels'cDNA constructs were subcloned into the pGEMHE plasmid vector. All cRNAs were transcribed using RibomaxTM Large Scale RNA Production Systems-SP6 and T7 Kit after linearizing the DNA constructs with appropriate restriction endonuclease. Oocytes injected desired cRNA were cultured in ND96 solution for 2³ days at 19²0℃environment. Whole cell KCNQ2/3 current was recorded using TEVC method. Results:â‘ The analysis of in vitro transcribed cRNA and agarose electrophoresis: The circular plasmid DNA showed more than one bands in the electrophoresis, while the linearized DNA showed only one band. The molecular of objective plasmid DNA was estimated with DNA marker. RNA has secondary structure and gave a smeared band on the gel. We used RNA sample buffer containing deionized formamide and formaldehyde to denature RNA, in order to get a clear RNA band on the eletrophoresis.â‘¡The electrophysiological characteristics of KCNQ2/3 current: KCNQ2/3 channel currents can be recoded using TEVC method at 2³ day after microinjection of Oocytes. The KCNQ2/3 current was elicited by a series of 2s depolarizing steps in 10mV increments from a holding potential of –80mV to +40mV and then return to the –60mV level. The characteristics of KCNQ2/3 current include low-threshlod, slow-activation, non-inactivation, voltage-dependent properties. At the different extracellular K+ solutions (25,50,75,96 mM), the reversal potential of KCNQ channel currents is consistent with the potassium equilibrium potential according the Nerst equation. Normalized tail current-voltage curve was fitted by Boltzmann function, and the half activation voltage (V_1/2), the minimal activation voltage and the slope constantk is –34.1±1.8mV, –60mV, and 9.6mV, respectively. The activation relaxation was fitted by single exponential function. The activation time constant (Ï„ac) became shorter following the increased depolarizing voltage steps. A typical deactivation relaxation was recorded with a protocol that has a prepulse of 20mV followed by a 1s test pulse to potentials from 0mV to –140mV. This relaxation can be fitted by two exponential functions.â‘¢The KCNQ2/3 potassium channel is less sensitive to the common potassium channel blockers such as 4-AP, TEA, Ba2+. TEA and Ba2+ inhibited KCNQ2/3 current in Oocyte with IC50 values of 3.1±0.54mM (n=0.92) and 0.77±0.17mM (n=0.83), respectively. 4-AP inhibited only 20% of KCNQ2/3 currents at the concentration of 4.5mM. On other hand, linopirdine is a potent blocker and fully inhibited KCNQ2/3 current at the concentration of 100μM. Conclusion: KCNQ2/3 potassium channel was expressed in Oocytes with methods of cRNA microinjection. The KCNQ2/3 current recorded using TEVC showed a low-threshlod, slow-activation, non-inactivating, voltage-dependent properties. This current is less sensitive to 4-AP, TEA,Ba2+, but is more sensitive to linopirdine and can be completely blocked by linopirdine at concentration of 100μM. 2 The regulation of the KCNQ potassium channel by extracellular pH. Aim: To explore the regulation of KCNQ potassium channel by extracellular pH. Methods: Oocytes injected with the cRNA of KCNQ2 and KCNQ3 potassium channel with 1:1 ratio were cultured in ND96 solution for 2³ days. Whole cell KCNQ currents were recorded using TEVC method and the effects of pH on the currents were observed. Results: â‘ Extracellular pH affected KCNQ2/3 currents in a concentration-dependent manner. Extracellular pH affected both the activation curve and the amplitude of KCNQ2/3 current. The decrease of pH value (increase H+ concentration) caused a depolarizing shift in the activation curve and a significant inhibition of the amplitude of KCNQ2/3 currents. The pH value that induced half-inhibition of KCNQ2/3 currents is 6.8. â‘¡Theregulation of KCNQ2/3 potassium channel by pH value was voltage-dependent. Lowering pH value slowed the activation of KCNQ2/3 currents, and the activation constant (Ï„) increased. Elevating pH value enhanced the activation of KCNQ2/3 currents, and the activation constant decreased. The effect of pH value on KCNQ2/3 current became smaller with more depolarized activation voltages. The effect of pH value was most significant at the more negative potential of around –60mV.â‘¢The effect of pH value on KCNQ2/3 current's deactivation. The slope of instantaneous current-voltage curve that indicates the permeation and conduction of the potassium channel became smaller in the low pH value solutions. These results suggest the KCNQ2/3 channel's activation and deactivation become slower in the more acidic solution. Conclusion: The KCNQ2/3 potassium channel was modulated by the extracellular pH value. Decrease of pH value leads to the inhibition of the amplitude of KCNQ2/3 currents, shifts activation curve to the right and slows de-or activation course. The pH value that causes half of maximal effect is 6.8. The profound effects of the extracellular pH exerted on KCNQ2/3 channel may play an important role during physiological neuronal activity and pathophysiological events such as epileptic seizures, cerebral ischemia and shock etc. 3 The regulation of the KCNQ potassium channel by extracellular K+ Aim: To explore the modulation of KCNQ2/3 potassium channel by extracellular K⁺. Methods: Oocytes injected with the cRNA of KCNQ2 and KCNQ3 potassium channel by 1:1 ratio were cultured in ND96 solution for 2³ days. Whole cell KCNQ currents were recorded using TEVC method and to observe in the effect of different concentrations of K⁺ solutions. Results:â‘ The KCNQ2/3 currents were enhanced by extracellular K⁺ in concentration-dependent manner. Elevation of external [K+] at a range of 0¹5mM potentiated KCNQ2/3 and KCNQ2 currents, and the effect of external [K+] was more significant on KCNQ2 currents than in KCNQ2/3currents. â‘¡Elevation of extracellular [K+] had no obvious effect on the activation curve of KCNQ2/3 channel. When extracellular [K+] was changed from 0 and 12mM, the initial potential of activation was not changed (–60mV), and the half activation voltage was shifted slightly to the left, and the slope of activation curve was reduced significantly from 11.2±0.4mV to 9.1±0.25mV.â‘¢Elevation of extracellular [K+] accelerated the activation of KCNQ2/3 currents in concentration-and voltage-dependent manner, so the process of the activation was enhanced more at more negative potential. â‘£Deactivation process of KCNQ2/3 channel became slow under the condition of elevated [K+]. Both fast and slow deactivation processes were prolonged significantly. Conclusion: Elevation of extracellular [K+], at the range of 15mM, potentiated KCNQ2/3 current in three aspects. First, it increased the amplitude of KCNQ2/3 currents in a voltage-dependent manner. Second, it accelerated the activation process and decelerated the deactivation process, and thus enhanced the function of the channel. Third, it didn't affect initial potential of activation, but significantly reduced the slope of activation. The effect of the above external [K+] is probably due to the increase of the conduction of KCNQ2/3 channels but not the number of available channels. 4 The regulation of KCNQ2/3 potassium channel by EGFR. Aim: To explore the regulation of KCNQ2/3 potassium channel by EGFR activation. Methods: The KCNQ2 and KCNQ3 potassium channel were co-expressed in HEK293 cell using Lipofectamine kits (Invitrogen). Whole-cell patch clamp recordings were made from HEK293 cell. The laser confocal microscopy technique was used to monitor the changes of membrane PIP2, PIP3 and intracellular Ca²⁺. Results: â‘ KCNQ2/3 currents were inhibited by long incubation with EGF in HEK293 cells. HEK293 cells expressing KCNQ2/3 channels were pre-incubated with EGF(100ng/ml) for 30 min. The KCNQ2/3 currents elicited from these cells were inhibited significantly; the amplitude of thecurrents was decreased to 28%±9.2(n=5,p<0.01); the half activation voltage was shifted from –16.6±1.2mV to -13.5±1.5mV; the activation and deactivation kinetic processes were slowed.â‘¡A tyrosine phosphatase inhibitor vanadate (100μM) mimicked EGF actions and decreased the KCNQ2/3 current to 45%±12.8(n=5,p<0.01); the time constant of activation and deactivation were prolonged from 230±37ms, 130±28ms to 535±42ms, 358±39ms, respectively.â‘¢The fast inhibition of KCNQ2/3 currents by activation of EGFR and M1 receptor was seen after a short application of EGF and acetylcholine. Both substances suppressed the KCNQ2/3 current reversible and the percentages of inhibition were 25%±5.3, 78%±10.8, respectively. â‘£The rapid suppression of KCNQ2/3 currents by EGFR and M1 receptor was attenuated by PLC inhibitor U73122 (3μM). The percentages of inhibition were 5%±4.3, 20%±5.7 by EGF and ACh, respectively in the presence of U73122. ⑤Wortmannin(3μM) and LiCl(100μmol/L) prevented the recovery of KCNQ2/3 current from the suppression by EGF and ACh. â‘¥PP2(200nM), a specific tyrosine kinase inhibitor, accelerated the recovery of KCNQ2/3 currents suppressed by EGF. ⑦The tyrosine phosphatase inhibitor vanadate (100μM), promoted the suppression and prevented the recovery of KCNQ2/3 currents. ⑧Activation of endogenous EGFR did not induce the PLCδ1PH-GFP and GRP1-PH–GFP translocation obviously, but induced the spark of intracellular Ca²⁺. Conclusion: â‘ EGF, when incubated with HEK293 cell for 30 min, inhibited expressed KCNQ2/3 currents, slowed the kinetics of channel. The effect of EGF on KCNQ2/3 channel was similar to the effect of Src kinase reported. The suppression could reversed by tyrosine kinase inhibitor PP2. Thus it is likely the result of phosphorylation action. â‘¡When EGF was applied to HEK293 cell expressing KCNQ2/3 currents, it also induced a rapid reversible suppression of the currents; the suppression of the current was attenuated by U73122; the recovery of the currents from the suppression was prevented by wortmannin and LiCl. These results suggested that hydrolysis of PIP2 probably underlied EGFR-mediated rapid regulation of KCNQ2/3currents. â‘¢Activation of endogenous EGFR receptor induced hydrolysis of PIP2, but did not induce an significant activation of PI3K.â‘£The regulation of KCNQ2/3 currents by EGFR activation went through two pathways: hydrolysis of PIP2, and phosphorylation. The increase of phosphorylation level probably facilitated the other receptor-mediated regulation of KCNQ2/3 currents. 5 The regulation of KCNQ2/3 potassium channel by GPCR Aim: To observe the regulation of KCNQ2/3 and M currents by activation of GPCR M1 and B2. To study the effect of oxo-M and bradykinin on the spike frequency of SCG neurons Methods: KCNQ channels and GPCR (M1 and B2) were expressed in CHO cell by transfection. Whole-cell patch clamp recordings were made from CHO and SCG cells to observe the effect of PMA on the regulations of potassium channel by M1 and B2 receptor activation. Current clamp recordings were made from SCG neuron to observe the modulation of spike frequency by M1 and B2 receptor activation. Results: â‘ the KCNQ2/3 current expressed in CHO cell was similar to the M-current recorded in SCG neuron, with characteristics of slow-activation and slow-deactivation kinetics. â‘¡The recovery of KCNQ2/3 current from the suppression by M1 receptor was faster than that of B2 receptor. â‘¢PMA(1μm) slowed the recovery of KCNQ2/3 current from the suppression by M1 receptor. â‘£The M-current recorded from SCG was inhibited by oxo-M and bradykinin, which leaded the membrane rest potential to depolarization and increased the spike frequency of neuron, suggesting that m-current played an important role in neuron excitability. Conclusion: The difference in the regulation of M-current by M1 and B2 receptor is probably due to the difference in the extent of phosphorylation, and increasing the level of phosphorylation perhaps enhanced the regulation of neural excitability by neurotransmitters.1. KCNQ2/3 potassium currents expressed in Xenopus oocytes, in HEK293 and CHO cells have similar characteristics as the M-currents recorded in SCG neurons. These characteristics include slow-activation and deactivation, non-inactivation, low-threshold activation and voltage-dependency. KCNQ2/3 currents were not very sensitive to the potassium channel blocker like TEA, 4-AP, Ba2+, but were sensitive to Linopirdine. 2. KCNQ2/3 current was regulated significantly by extracellular pH. Decrease of the extracellular pH value caused the inhibition of the KCNQ2/3 current; the amplitude of the currents were suppressed; the activation curve was shifted to the right; the activation and deactivation kinetics were slowed. The effects of pH were voltage-and concentration-dependent, with the effect being most significant at the potentials near the threshold for activation. The effect of extralcellular pH was greater on KCNQ2/3 currents than on KCNQ2 currents. 3. Elevation of extracellular [K+], at the range of 0-15mM, potentiated KCNQ2/3 current in three aspects. First, it increased the amplitude of KCNQ2/3 currents, and this effect was more significant at positive voltages. Second, it accelerated the activation process and decelerated the deactivation process, an effect probably through acting on the state of open probability and thus enhanced the function of channel. Third, it didn't affect threshold potential for activation, but affected the slope significantly. Elevation of external [K+] appeared to increase the conduction of KCNQ2/3 channels but not the number of available channels. 4. Activation of endogenous EGFR of HEK293 regulated the KCNQ2/3 currents in two distinct mechanisms: rapid reversible inhibition resulted from hydrolysis of PIP2,and slow inhibition realized probably by phosphorylation. The pathway of PI3K seemed not involved in the effect of EGFR activation. The effects of EGFR and M1 receptors were enhanced using the PTP inhibitor. These results suggested that the hydrolysis of PIP2 and phosphorylation were two important mechanisms involved regulation of KCNQ2/3 currents by the receptor activation. Phosphorylation action might enforce the inhibition...