Molecular Mechanism For Membrane Depolarization Induced Potential Of KCNQ2/Q3 Currents

Potassium channels selectively permit K+ go through, and have the largest family of all ion channels with the most complex functions in various physiological functions. They are widely distributed in the cells of skeletal muscle, nerve, blood vessels, trachea, and gastrointestinal tract. Potassium channels play an important role in the regulation of cell membrane potential and excitability as well as smooth muscle activity. The outward delayed rectifier potassium ion channel encoded by KCNQ gene has six transmembrane domains and a pore. Two members of this family, KCNQ2/3 are the molecular basis of the neuronal M currents that play a critical role in neuron excitability. These channels have slowly activating, non-inactivating, voltage-dependent potassium currents.M currents were named because of its suppression by muscarinic receptor activation. Many neurotransmitters or hormones, such as ATP, bradykinin, angiotensin II, substance P and Luteinizing hormone releasing hormone have also been shown to be able to inhibit M currents. M currents are widely distributed in peripheral as well as central neurons. M currents play a critical role in the determination of neuronal excitability. Blocking of M currents will result in hyper excitability of the neuronal system. The dysfunction of M channel is closely associated with diseases like benign neonatal familial convulsions (BFNCs), Alzheimer, epilepsy, and other diseases. Ample evidence demonstrated that activation of some Gq protein-coupled-receptors will result in KCNQ/M current inhibition. Membrane PtdIns (4, 5) P2 (PIP2) hydrolysis and channel phosphorylation are two mechanisms that have been proposed for modulation of KCNQ2/3 currents. Phospholipids C (PLC) hydrolyzes PIP2, yielding two intracellular second messengers IP3 and DAG. IP3 then trigger calcium release from endoplasmic reticulum, which in turn are involved in varieties of cell functions. DAG activates PKC. A steady level of PIP2 is maintained by the hydrolysis and the synthesis of PIP2 and for the latter PI4K plays an important role.We found that the cell membrane depolarization could increase KCNQ2/3 currents expressed in Xenopus oocytes. However, the underlying mechanism was unknown. We hypothesized that membrane PIP2 level and/or channel phosphorylation may involved in the membrane depolarization-induced potentiation of KCNQ2/Q3 currents.Objective: To study the possible involvement of channel phosphorylation and membrane PIP2 metabolism in membrane depolarization-induced potentiation of KCNQ2/Q3 currents.Methods: (1) Transcription of the KCNQ2/3 channels in vitro: cDNA coding KCNQ2/3 was inserted into the pGEMHE plasmid vector and the sequence were confirmed by sequencing. All cRNAs were transcribed using RibomaxTM Large Scale RNA Production Systems T7 Kit after linearizing the DNA constructs with appropriate restriction endonuclease NheI. (2) Oocytes were surgically isolated from anesthetized adult female frogs (Xenopus laevis) and treated with 2 mg/ml collagenase (Type II, Sigma) in the OR2 solution (in mM, 82 NaCl, 2 KCl, 1 MgCl2, 5 HEPES, pH 7.4) for 90 min at room temperature (~25°C). After three washes with OR2 solution, the oocytes were incubated at 18°C in ND96 solution (in mM, 96 NaCl, 1 KCl, 1 MgCl2, 1.8 CaCl2, 5 HEPES, pH 7.4). cRNA was injected in oocyte. (3) Currents were measured from oocytes 2-3 days after cRNA injection under two-electrode voltage clamp using 0.5-1.0 M? microelectrodes filled with 3 M KCl (PH 7.2) with a Geneclamp 500B amplifier (Axon Instruments). PMA was applied in the bath solution of ND96. (4) Membrane protein extraction and Western blotting. The injected or uninjected oocytes were lysed with a glass homogenizer. Homogenate was spun at 5000 g for 5 min at 4°C and the resulting supernatant was further spun at 200,000 g for 60 min. The pellet containing the membrane fraction was resuspended in the lysis buffer (1 nl/oocyte). The membrane proteins were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Standard Western blottings, using Tris-buffered saline (TBS) and 5% non fat milk powder were performed. Special primary antibody and IR dye680-conjugated secondary antibody were used. All washing steps were performed using TBST. Blots were then scanned using the Odyssey Infrared Imaging System (LiCor, Lincoln, NE). Immunoprecipitation: The pellet containing the membrane fractions were resuspended in the lysis buffer, Special primary antibody was added. After rotating at 4°C overnight, protein G beads were added. The beads were washed and western blottings were performed. (5) 100 oocytes from either control group or RNA injected group (2-3 days after injection) were used for western blots and immunoprecipitation studies. Oocytes were solubilized in 500μl of lysis buffer on ice. The lysis buffer contained (in mM): 5 Tris-HCl, 1 EDTA, 1 EGTA, 10 Na3VO4, 10 NaF, and (inμg/ml) 30 PMSF, 10 pepstatin A, 2.5 leupeptin, 10 aprotinin. Homogenates were centrifuged at 400 rpm for 10 min at 4°C to remove yolk granules. The supernatant was centrifuged at 12,000 g for 30min at 4°C, yielding a whole cell protein supernatant. Western blottings were performed. (6) Immunocytochemistry. Immediately after pretreatment with PMA or ND96-K , oocytes expressed KCNQ2/3 channels were fixed in 4% paraformaldehyde, and then were placed in blocking buffer containing 3% BSA and 0.2% tritonX-100, PBS. Special primary antibodies were added overnight. Fluorescein isothiocyanate secondary antibodies were used. The cells were then scanned using the laser scanning confocal microscopy. (7) Immunohistochemistry. Oocytes were fixed and paraffin sections were made. Paraffin sections were deparaffinized in toluene and rehydrated in decreasing concentrations of alcohol. Oocytes sections were then boiled for 5 min in 0.1 M citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by treatment in H2O2 for 10 min. Nonspecific binding was then blocked in PBS/10% BSA for 1hr. Special primary antibody were added overnight. Fluorescein isothiocyanate secondary antibodies were used. The cells were then scanned using the laser scanning confocal microscopy. (8)RT-PCR. Total RNA was extracted from different groups of oocytes and concentrations were adjusted to 800 ng/μl. RNA was reversely transcribed into DNA and the latter was used as a template to amplify PI4K with PI4K-specific primer, and the resulted PCR product indicates the mRNA level of PI4K. (9) RNAi. Double strands RNA (dsRNA) or siRNA interference (RNAi) against endogenous PI4 kianse of Xenopus oocytes (PI4K) was used to depress the enzyme. The 646 base (corresponding to base 1038-1671) pairs dsRNA was synthesized from a cDNA clone of PI4K isolated from Xenopus oocytes (ordered from Open Biosystems, USA. Genebank access no. BC073760) by PCR using a ProofStart PCR kit, and was used as the template for transcription of dsRNA or ssRNA. The templates for siRNA or ssRNA were synthesized chemically. The long dsRNA or ssRNA and siRNA were transcribed from the templates using a T7 RiboMAX? Express RNAi System (Promega Corporation Madison, WI 53711-5399 USA). The transcripts were assessed for integrity on a 1.5% agarose gel or 20% PAGE gel, and diluted in injection buffer (0.1-0.2mg/ml) and stored at–80oC until use. (10) PKC activity of Xenopus oocyte was measured by kit of PepTag Assay for Non-Radioactive Detection of Protein Kinase C (Promega Corporation Madison, WI 53711-5399 USA). 100-200 oocytes were homogenized in 1 ml of cold PKC extraction buffer. The lysate was centrifuged at 14,000 g for 5 minutes, 4°C, and the supernatant was saved and used for the PKC assay according to the supplier's protocol.Results: (1) Molecular biology. A proper restriction enzyme was chosen to cut the circular plasmid and the linearized plasmid DNA were analysed by agarose electrophoresis. The circular plasmid DNA showed more than one bands in the electrophoresis, while the linearized DNA showed only one band. The band of cRNA in vitro transcribed is uniform, clean, and no sign of degradation. (2) Expression of KCNQ2/Q3 currents. After microinjection of KCNQ2 and KCNQ3 RNAs into oocytes for 2-3 days, KCNQ2/Q3 currents could be recorded by two-electrode voltage clamp method. A ramp voltage-clamp protocol from -80 mV to +40 mV was used to evoke KCNQ2/Q3 currents. Results of western blot also showed the expression of KCNQ2 and KCNQ3 channel proteins. (3) Immunoprecipitation results indicated that the phosphorylation status of KCNQ2 and KCNQ3 proteins was not changed by membrane depolarization (high K+ solution); the quantified phosphorylation intensity values for control and depolarization-treated groups were 1.01±0.02 (n = 6) and 0.99 ±0.03 (n = 5), respectively. (4) Depolarization (high K+) and PMA incubation increased PI4 kinase expression in oocytes as detected by western blot, immunofluorescence and immunocytochemistry studies. The time course of PI4 kinase expression was also studied. The expression of PI4 kinase detected by western blot began to increase when the oocytes were incubated in ND96K solution for 5 min, and continue to increase during the period of 20 min observation, reaching 1.88±0.19 of the control (0 min) at the time of 20 min. (5) Knocking down the PI4 kinase expression prevented the effect of the depolarization on PI4 kinase expression and eventually on enhancement of KCNQ2/Q3 currents. Injection of the dsRNA reduced the basal expression level and abolished the depolarization-induced increase of PI4 kinase expression, as demonstrated by western blot. These results were confirmed by the immunofluorescence experiment. In agreement with these results, dsRNA completely abolished the depolarization-induced enhancement of KCNQ2/Q3 currents. siRNA had a similarly effect, but not the ssRNA(sramble small RNA). (6) PKC blockers bisindolylmaleimide (Bis) was tested for its effects on the depolarization- and PMA-induced increase of PI4 kinase expression. Bis blocked both increases. (7) In PKC activity assay, both the depolarization and PMA increased the activity of PKC. On other hand, PKC expression was not altered by the depolarization or PMA. (8) Maneuvers that inhibit intracellular Ca2+ rising (Ca2+-free plus EDTA, Cd2+, thapsigagin, ionomycin) did not alter the depolarization-induced potentiation of KCNQ2/Q3 currents.Conclusion: (1) Membrane depolarization did not change the phosphorylation status of KCNQ2/Q3 potassium channels, which is consistent with pharmacological results. Channel phosphorylation should not be the mechanism for depolarization-induced potentiation of KCNQ2/Q3 currents expressed in Xenopus oocytes. (2) PIP2 is known to be able to activate KCNQ2/Q3 current. The present experimental results showed that membrane depolarization can activate PI4K, which would result in an increased synthesis of PIP2. (3) PMA, an activator of PKC, not only enhanced KCNQ2/Q3 currents, but also increased activity of PI4K. (4) Membrane depolarization can increase PKC activity. (5) Bis, a PKC inhibitor, blocked PI4K activity increase induced by the membrane depolarization. The above results indicate that the membrane depolarization-induced potentiation of KCNQ2/Q3 currents is the result of sequential effects of PKC activity increase, enhancement of PI4K activity and increased synthesis of PIP2.