Study On Membrane Depolarization-induced Increases Of KCNQ2/Q3 Currents And Membrane PIP₂ Level

Growing body of evidences show membrane phosphatidylinostol 4,5-bisphosphates (PtdIns(4,5)P2, PIP2) plays important role in cell signaling. Presence of PIP2 is fundamentally important for maintaining function of a large number of ion channels and transporters, and for other cell processes such as vesicle trafficking, mobility and endo- and exocytosis. Membrane PIP2 is not only the precursor of some signaling molecules such as diacylglycerol (DAG) and inositol trisphosphate (IP3), PIP2 is a signaling molecule in its own right and its level in the membrane is dynamically modulated. A major metabolic modulation of membrane PIP2 level is phospholipase C (PLC)-mediated PIP2 hydrolysis. This event related mechanism has been contributed to be the underlying mechanism for receptor-induced modulation of function of ion channels. The membrane PIP2 level is maintained by many phosphoinositides kinases and phosphotases. In this study, we describe a novel mechanism of membrane PIP2 modulation. Membrane depolarization induces elevation of membrane PIP2 and subsequently increases KCNQ2/Q3 currents expressed in Xenopus oocytes. Further evidence suggests the depolarization-induced elevation of membrane PIP2 is through activation of PKC and increased activity of PI4 kinase.1.The effect of membrane depolarization on KCNQ/Q3 current expressed in Xenopus oocytes.Objective: In previous studies, we noticed that KCNQ2/Q3 current expressed in Xenopus oocytes was sensitive to membrane potential, in a manner different from classical voltage-dependent gating of channels. In this part of the study, we studied this phenomenon in details, and investigated possible underlying molecular mechanisms.Methods: Plasmids encoding KCNQ and Kir were linearized and cRNA were synthesized by in vitro transcription, and expressed in Xenopus oocytes, Two electrodes voltage clamp (TEVC) recording was used to measure the modulation of depolarization and extracellular K+ on KCNQ currents.Results: (1) KCNQ2/Q3 currents expressed in Xenopus oocytes could be increased by a continuous depolarization (-40 mV~ +40 mV) with the characteristics of time- and voltage-dependency. When the membrane was repolarized back to -80 mV, the depolarization-induced increase was reversed, indicating that voltage-dependent current increase is reversible. The increased current could be inhibited by linopirdine (30μM), a specific M/KCNQ blocker, indicating that the increased current is KCNQ2/Q3 current. (2) Depolarization did not affect the activation and deactivation kinetics of KCNQ2/Q3 currents when the activation was measured at 0 mV and deactivation was measured at -60 mV either before or after a 0 mV depolarization for 15 min. Similarly, the conductance-voltage relationship of KCNQ2/Q3 activation was not affected by the depolarization. (3) Subunit specificity for effects of depolarization (0 mV, 15 min) was studied. Depolarization could augment KCNQ2 and KCNQ2/Q3 currents, but not KCNQ1 or KCNQ1/KCNE1 currents. The pattern of effects of depolarization on KCNQ2 current is similar to that on KCNQ2/Q3. The half-maximum increase voltages (V1/2) for KCNQ2 and KCNQ2/Q3 were similar. Similar to KCNQ2/Q3, depolarization did not affect the kinetics of KCNQ2 currents. (4) Kir2.1 and Kir2.3 channels were expressed in Xenopus oocytes to study the effects of depolarization (+40 mV) on these channels. Both Kir currents were increased but Kir 2.3 currents were increased by depolarization do greater degree than Kir2.1 currents. This result is consistent with our earlier study that indicates Kir2.1 channel has higher affinity with PIP2 than Kir2.3. This result is also in accordance with the notion that modulation of currents by depolarization may be a result of increased PIP2 level. (5) We tested the effect of external K+ on KCNQ2/Q3 currents to exclude the possibility that the depolarization-induced enhancement of KCNQ2/Q3 currents is due to an increased out flux of K+ and increased concentration of external K+. Increasing external K+ to 10 mM did not affect depolarization-induced potentiation of KCNQ2/Q3 currents. (6) High external K+ solution (ND96K: 96 mM K+) was used to depolarize the membrane to mimic the effect of voltage-clamped depolarization. ND96K incubation (membrane depolarization) also led to enhancement of KCNQ2/Q3 currents. The average folds of increase by ND96K incubation were not significantly different from the voltage-clamped experiments (0 mV, 15 min). The results suggest the depolarization per se contributes to the observed enhancement of KCNQ2/Q3 currents.Conclusions: (1) Membrane depolarization augments amplitude of KCNQ2/Q3 currents expressed in Xenopus oocytes with the characteristic of time- and voltage-dependency. (2) Depolarization did not affect he activation and deactivation kinetics and the conductance-voltage relationship of KCNQ2/Q3 current. (3) Depolarization-induced increase of KCNQ currents is subunits sensitive. Depolarization had no effects on KCNQ1 and KCNQ1/KCNE1. (4) Kir2.1 and Kir2.3 currents were also sensitive to depolarization. However depolarization increases Kir2.1 and Kir2.3 current in a manner relating to their affinity to PIP2. (5) An increase of external K+ by depolarization could not be causal factor for potentiation of KCNQ2/Q3 currents. (6) External high K+ could mimic the effect of membrane depolarization in increasing KCNQ2/Q3 currents and in a similar manner in regarding the effect of kinetics and voltage-dependent activation of KCNQ2/Q3 currents. The results strongly suggest the depolarization per se contributes to the observed enhancement of KCNQ2/Q3 currents. 2. The mechanism of depolarization-induced elevation of PIP2 level and augment of KCNQ2/Q3 current.Objective: The first part of the study demonstrated that depolarization induced KCNQ2/Q3 current and an elevation of PIP2 level may be involved. We focused our study of this part on the underlying molecular mechanism.Methods: (1) Ci-VSP was co-expressed with KCNQ2/Q3 in Xenopus oocytes, and TEVC recordings were used to study the modulation of KCNQ2/Q3 current and PIP2 level by activation of Ci-VSP and depolarization. (2) Blocker of PI4 kinase was used to test whether PI4 kinase was involved in the depolarization-induced PIP2 elevation. (3) Activator and blocker of PKC were used to test whether PKC was involved in the depolarization-induced PIP2 elevation. (4) TLC method was used to measure phosphoinositides level directly to study the effect of depolarization and activation of PKC on PIP2 level. (5) Blocker of PKA was used to test whether PKA was involved in the depolarization-induced PIP2 elevation. (6) Perforated patch clamp was used to study the effect of depolarization on M current in DRG neurons and KCNQ2/3 currents expressed in CHO cells.Results: (1) Depolarization-induced potentiation of KCNQ2/Q3 currents can be prevented and reversed by activation Ci-VSP, indicating an involvement of PIP2. Measurement of PIP2 using TLC method directly demonstrated that depolarization increased PIP and PIP2 level. (2) Hypertonic stress increases PIP2 level by activating PIP5KIβ. If our depolarization-induced increase of KCNQ2/Q3 currents depends on extra PIP2 synthesis, pre-incubating the oocytes with hypertonic solution should blunt the increase. The result shows that depolarization-induced increase was significantly reduced by hypertonic stress. (3) Wortmannin, which inhibits PI4 kinase, greatly reduced the depolarization-induced enhancement of KCNQ2/Q3 currents. (4) Activation of PKC by PMA increased KCNQ2/Q3 current in a similar manner as depolarization, and similarly had no effect on KCNQ1 and KCNQ1/KCNE1. PMA also increased PIP and PIP2 level measured by TLC. (5) H-89, a inhibitor of PKA, could not inhibit depolarization-induced KCNQ2/Q3 current increase, indicating that PKA is not involved in depolarization-induced potentiation of KCNQ2/Q3 currents. (6) Depolarization did not augument M current in DRG neurons and KCNQ2/3 currents expressed in CHO cells; Activation of PKC inhibited KCNQ2/3 currents expressed in CHO cells. Large depolarization (+20 mV) inhibited M current in DRG neurons reversibly.Conclusions: (1) The membrane depolarization increases KCNQ2/Q3 currents through increasing membrane PIP2 level. (2) Pre-incubating the oocytes with hypertonic solution blunt the depolarization-induced KCNQ2/Q3 current increase. (3) The depolarization increases PIP2 level through activation of PI4 kinase. (4) Activation of PKC mediates the depolarization- induced KCNQ2/Q3 current increase. (5) PKA is not involved in the depolarization-induced KCNQ2/Q3 current increase. (6) Depolarization do not affect M current in DRG neurons and KCNQ2/3 currents expressed in CHO cells. Activation of PKC inhibits KCNQ2/3 currents expressed in CHO cells.