| Inwardly Rectifying K+ Channels (Kir) are a group of potassium channels extensively distributed in many kinds of tissues. It is known for its inwardly rectifying property. Kir2.0 is characteristic for its strong inward rectifying property. This property of Kir2.0 confers it the role to help to maintain the resting membrane potential of the cell by keeping the membrane potential near to the K+ equilibrium potential. Kir 2.0 is also involved in the process of repolarization of membrane action potential. Kir2.3 is an important member of Kir2.0 family. It shares the common characters of inward rectifying potassium channel: (1) K+ influx is easier than its efflux. (2) The channel function depends on membrane phospholipid—specifically PIP2. (3) The channel is targets of modulation of many types of factors. (4) All Kir channels share the same basic structure. The N-terminus of the channel starts from the interior of the cell, and after twice transmembrane foldings, the C-terminus also ends in the interior of the cell.It is known that PKC activation can lead to the inhibition of some Kir channel (Kir2.3,Kir3.x and Kir6.x) functions. PKC is a Ser/Thr protein kinase whose activation depends on calcium, phospholipid and diacylglycerol. PMA is an activator of PKC. It has been reported that PMA has an inhibitory effect on some Kir channels. However, the effects of PMA on Kir channels are variable depending on the channel type studied or the tissues they expressed. It has been reported that PMA inhibits Kir2.3 channel, and that Kir2.1 is not sensitive to PMA. Kir2.3 channels are very sensitive to the inhibitory effect of PMA when it is expressed in Xenopus oocytes. However no inhibition can be seen when Kir2.3 channels are expressed in CHO cells. Thus although there have some reports about PMA inhibition of Kir2.3, the underlying mechanism is not fully elucidated. This study will focus on the mechanism of PMA action on Kir2.3 channels. We also studied the effects of genistein on Kir2.3 channels. Genistein is a widely used inhibitor of protein tyrosine kinase. When we studied the tyrosine phosphorylation of Kir2.3 channels, we found that genistein inhibited Kir2.3 currents. We decided to have a through investigation into the mechanism of genistein action.1. PMA inhibited Kir2.3 currents through PKC pathway.Aim: To express Kir2.3 channels in Xenopus oocyte and study the properties of Kir2.3 channels. To study the effects of PMA, an activator of PKC, on Kir2.3 currents.Methods: (1) Transcription of Kir2.3 channels in vitro. cDNA coding Kir2.3 was inserted into the pGEMHE vector. The cRNA was transcribed after linearizing the DNA constructs. (2) Preparation and microinjection of oocytes. Oocytes from adult female frogs were used. Xenopus oocytes were treated with 2 mg/ml collagenase (Type II, Sigma) in the OR2 solution for 90 min. After washes with the OR2 solution, the oocytes were incubated at 18°C in the ND96 solution and injected. (3) Currents recording. Whole cell currents were recorded under two-electrode voltage clamp. PMA was applied in the bath solution of ND96K.Results: (1) Molecular biology. A restriction enzyme was chosen to cut the circular plasmid and the linearized plasmid DNA were analyzed by agarose electrophoresis. The plasmid DNA showed more than one bands in the electrophoresis, whereas the linearized DNA showed only one band. The band of cRNA in vitro transcribed is uniform, clean, and has no sign of degradation. (2) The property of Kir2.3 currents. Kir currents can be recorded using two-electrode voltage clamp method. A series of ramp voltage-clamp from -90 mV to +90 mV was used to evoke the Kir2.3 currents. Oocytes were constantly perfused with ND96K. ND96 was used to reduce most of the Kir currents at -80 mV. The current-voltage relationship (I-V) curve of Kir2.3 channel currents showed the significant inward rectifier property. When ND96K solution was used, the reversal potential of Kir2.3 currents was around 0 mV. Ba2+ blocked these currents effectively. (3) PMA inhibited Kir2.3 currents. Bath application of 100 nM PMA inhibited the currents amplitude by 46.3±5.2%. The inhibition developed relatively slowly with a delay for 2-5 min, and reached a stable level within 5-10 min. Upon washing out of PMA for 10-20 min, the Kir2.3 currents showed no sign of recovery. PMA did not alter the rectification property of Kir2.3 currents. PMA concentration-dependently inhibited the currents of Kir2.3 channels expressed in Xenopus oocytes with an IC50 of 17.7±0.13 nM. (4) PMA inhibited Kir2.3 currents through PKC pathway. PDBu, another activator of PKC, also inhibited the Kir2.3 currents by 40.8±3.4%; 4α-PMA was an inactive analog of PMA and had no inhibition on the channel currents. Bisindolylmalimide, a PKC blocker, almost abolished the PMA effect. These results showed that PMA affected Kir2.3 currents by PKC activation.Conclusion: Kir2.3 channels could be expressed in Xenopus oocytes by microinjecting the transcribed cRNA. By using two-electrode voltage clamp, the currents through Kir2.3 could be observed with the characteristics of inwardly rectification. Ba2+ blocked Kir2.3 currents. PMA, an activator of PKC, concentration-dependently inhibited the currents of Kir2.3 channels. PKC blocker prevented the effect of PMA.2. The study on the mechanism of PKC inhibition of Kir2.3 currents.Aim: To explore the mechanism of PKC inhibition of Kir2.3 currents by the methods of molecular biology and electrophysiology.Methods: (1) Molecular biology. cDNA coding Kir2.3, Kir2.1 and the mutants was inserted into the pGEMHE vector. Chimeric constructs were prepared by the overlap extension polymerase chain reaction. Site-specific mutants were produced with a QuickChange kit (Stratagene, La Jolla, CA). Orientation of the constructs and correct mutants were confirmed with DNA sequencing. (2) Membrane protein extraction and Western blotting. The injected or uninjected oocytes were lysed with a 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 (1μl/oocyte). The membrane protein were resolved by 10% SDS-PAGE and transferred to PVDF membranes. Standard Western blottings were performed. Specific primary antibody and IR dye680-conjugated secondary antibody were used. Blots were then scanned using the Odyssey Infrared Imaging System (LiCor, Lincoln, NE). (3) Detection of proteins on the surface of oocytes. Immediately after pretreament with PMA, oocytes expressing Kir2.3 channels were fixed in 4% paraformaldehyde, and incubated with NHS-SS-biotin at 4°C overnight. After being homogenized and centrifuged, Neutravidin-linked beads were added to the supernatant. The beads were washed and western blottings were performed. (4) Immunoprecipitation. The pellet was resuspended in the lysis buffer, and the primary antibodies were added. After rotating at 4°C overnight, protein G beads were added. After washing the beads, western blottings were performed. (5) Immunocytochemistry. Immediately after pretreament with PMA, oocytes expressing Kir2.3 channels were fixed in 4% paraformaldehyde, and were placed in blocking buffer. The primary antibodies were added and incubated overnight. Fluorescein isothiocyanate secondary antibody was used to recognize the primary antibody and visualized using the laser scanning confocal microscopy. (6) autoradiography. Oocytes expressing Kir2.3 channels were incubated with 32P (0.5 mCi/ml) overnight. After pretreament with PMA, the membrane protein were extracted. Kir2.3 protein were immunoprecipited, and transferred to PVDF membrane. The membranes were subjected to autoradiography using Kodak film at -80°C. (7) Electrophysiology: The currents were record as described above.Results: (1) Characteristic interactions with PIP2 determined the inhibition of Kir2.3 currents by PKC. 100 nM PMA inhibited the currents of Kir2.3 channels expressed in Xenopus oocytes by 46.3±5.2%, whereas Kir2.1 is not sensitive to PMA inhibition. PMA had a weaker inhibition on Kir2.3(I213L) of 16.9±3.1% than on Kir2.3 and a stronger inhibition on Kir2.1(L222I) of 8.9±1.7% than on Kir2.1. It had been shown that Kir2.3 had a weaker interaction with PIP2 than Kir2.1. Kir2.3(I213L) is a mutant that has a stronger interaction with PIP2 and Kir2.1(L222I) is a mutant that has a weaker interaction with PIP2. So the characteristic interactions with PIP2 determined the modulation of Kir2.0 channels by PKC. (2) T53 and I213 were both important for PMA-induced inhibition of Kir2.3 currents. Amino acid sequences of Kir2.3 shared high homology of 58% with those of Kir2.1, which suggests that recombinant channel proteins were likely to produce functional channels. According to the widely accepted transmembrane topology, both Kir2.3 and Kir2.1 channels have their N and C terminus inside the membrane, two transmembrane-folding domains (M1 and M2) and putative pore regions (P). Chimeras were named based on the following rules: numbers 1 and 3 indicated Kir2.1 and Kir2.3, respectively, letter N and C represented N and C terminus, respectively, and the P letter for the rest sequence (transmembrane domains, M1 and M2, and pore region). 100 nM PMA inhibited N3P1C3 by 42.8±3.8% and had no effect on N1P3C1. This result indicated that the N and C termini distinguished the difference of Kir2.3 and Kir2.1 on their sensitivity to PMA inhibition. PMA inhibited N1P3C3 by 12.1±2.0% and N3P1C1 by 33.6±2.5%; PMA inhibited N3P3C1 by 25.3±4.1% and N1P1C3 by 13.9±2.7%, Thus both the N and C termini are essential for a full sensitivity of Kir2.3 to PMA regulation. To further find the sites of PKC action, we constructed two mutants: Kir2.3(T53I) and Kir2.1(I79T). These mutations had been shown to be important for PMA inhibition of Kir channels. PMA inhibited Kir2.3(T53I) by 36.3±3.1%, which was significantly different from PMA inhibition on Kir2.3 (p<0.05). PMA did not inhibit Kir2.1(I79T). As it has been showen, I213 was also important for PMA inhibition on Kir2.3 currents. We constructed two other mutants: Kir2.3(T53I, I213L) and Kir2.1(I79T, L222I). Kir2.3(T53I, I213L) was almost fully avoid of PMA inhibition, with an inhibition of only 4.2±9.3%, which was not significantly different from PMA inhibition on Kir2.1 (p>0.05). PMA inhibited Kir2.1(I79T,L222I) by 13.8±1.5%, which was significantly different from PMA inhibition on Kir2.1 (p<0.05). Thus both the T53 and I213 are essential for a full action of PMA on Kir2.3. (3) T53 and I213 in Kir2.3 are also the key amino acids in determining the modulation of Kir2.3 by other factors. As we reported before, a: azide and KHCO3 both could lower the intracellular pH and inhibit Kir2.3 currents, but had not effects on Kir2.1 currents. b: wortmannin (a widely used PI-4K inhibitor and thus could block the synthesis of PIP2) inhibited potassium channel currents and the degree of the inhibition were negatively correlated with PIP2-channel interactions. We further tested the effects of azide, KHCO3 and wortmannin on Kir2.1, Kir2.3 and their mutants and compared the results with those of the inhibiton effects of PMA on these channels. Azide, KHCO3 and wortmannin all inhibited the currents through Kir2.3(T53I), Kir2.3(I213L) and Kir2.3(T53I, I213L) to a less degree than on the currents through Kir2.3; on the other hand, azide, KHCO3 and wortmannin all inhibited the currents through Kir2.1(79T), Kir2.1(L222I) and Kir2.1(I79T, L222I) to a greater degree than on the currents through Kir2.1. Based on the above results from (2) and (3), we believe that like other modulators, PMA possibly inhibits Kir2.3 currents through an alteration of channel-PIP2 interaction. (4) Kir2.3 channels were not phosphorylated under the condition of PMA treatment. We found that N terminus was more important for PMA inhibition. There were only two possible phosphorylation sites: S36, S39. We made a mutant Kir2.3 (S36G, S39G) and found that PMA could inhibit Kir2.3 (S36G, S39G) by 56.0±2.6%, which was not differet from inhibion of Kir2.3 Okadaic acid (OA) was a phosphatase inhibitor, and it did not change the inhibition effect of PMA on Kir2.3 currents in our study. We also directly studied the phosphorylation state of Kir2.3 channels. The results of autoradiograph showed that Kir2.3 channels were not phosphorylated directly in vivo by PMA. On the other hand, PKC, as a positive control, was autophosphorylated by treatment with PMA. Thus the results showed that activation of PKC by PMA also phosphorylate PKC itself but did not phosphorylate Kir2.3 channels. (5) The protein interlization might be involved in the inhibition of Kir2.3 currents by PKC. Phalloidin's toxicity is attributed to its ability to bind to F actin and prevented its depolymerization. Latrunculin A is an inhibitor of the microfilament-mediated processes of fertilization and early development and it can disrupt microfilament-mediated processes. We tested the effects of phalloidin and Latrunculin A. After application of phalloidin (PLD), PMA inhibited Kir2.3 by 7.5±1.6%; After application of Latrunculin A (LAT), PMA inhibited Kir2.3 by 31.3±3.3%. Thus both phalloidin and latrunculin A reduced the inhibitory effect of PMA on Kir2.3 currents. We also used confocal microscopy to examine the effect of PMA on the Kir2.3 surface expression. Quantitation of the images shows that the immunofluorescence in the oocytes expressing Kir2.3 significantly decreased to 51.1±8.5% after PMA treatment. For the oocytes expressing Kir2.1, the immunofluorescence was not changed. We directly measured the changes in surface expression of Kir2.3 channels in oocytes using Western blots by labeling cell-surface proteins with NHS-SS-biotin. After PMA treament, PMA significantly decreased the surface protein of Kir2.3 to 61.2±10.4%.Conclusion: Characteristic interactions with PIP2 determined modulation of Kir2.0 channels by PKC. Like other modulators, PMA possibly inhibited Kir2.3 currents through an alteration of channel-PIP2 interaction. Kir2.3 channels were not phosphorylated under the condition of PMA treatment. The protein internalization might be involved in PKC inhibition of Kir2.3 currents. The characteristic interactions between Kir channel-PIP2 might also determine the internalization of Kir channels.3. Molecular basis for genistein-induced inhibition of Kir2.3 currents.Aim: To study the effect of genistein, an inhibitor of protein tyrosine kinase, on Kir2.3 and Kir2.1 channels expressed in Xenopus oocytes and HEK293 cells.Methods: (1) Molecular biology: Kir2.3, Kir2.1 and the mutants were expressed as described above. (2) Membrane protein extraction, Western blotting and immunoprecipitation were done as described above. (3) Electrophysiology: Two-electrode voltage clamp were used to record the currents in Xenopus oocytes. The Kir2.3 and Kir2.1 channels were expressed in HEK293 cells using Lipofectamine kits (Invitrogen). Whole-cell patch clamp recordings were made from HEK293 cells.Results: (1) Genistein significantly inhibited Kir2.3 currents. In this study, we found that among three members of the Kir family (Kir2.3, Kir2.1, and Kir3.4* [a highly active mutant of Kir3.4, Kir3.4-S143T]) we tested, genistein significantly inhibited Kir2.3 currents. Using the two-electrode voltage clamp technique, we have demonstrated that micromole concentrations of genistein concentration-dependently and reversibly inhibited the currents of Kir2.3 channel expressed in Xenopus oocytes with an IC50 of 16.9±2.8μM. 100μM genistein inhibited Kir2.3 currents by 44.8±2.1%. Using the whole-cell patch-clamp technique, genistein inhibited the currents of Kir2.3 channel expressed in HEK293 cells with an IC50 of 19.3±3.2μM. In Xenopus oocytes, the inhibitory effect developed relatively fast and reached a stable level after 90.7±4.2 s and could be washed out easily. (2) The inhibitory effect of genistein on Kir2.3 currents did not involve an alteration of protein tyrosine phosphorylation. The effect of genistein on Kir2.3 currents was not affected by vanadate, a potent protein tyrosine phosphatase inhibitor. Furthermore, the effect of genistein was not mimicked by daidzein, an inactive analogue of genistein, or another potent tyrosine kinase inhibitor, tyrphostin 23. In immunoprecipitation experiments, a tyrosine phosphorylation state of Kir2.3 channels was not detected by anti-phosphotyrosine antibody PY99. (3) Channel-PIP2 interactions were not involved in genistein inhibition of Kir2.3 currents. Kir2.3(I213L) is a mutant that has a stronger interaction with PIP2. Genistein at 100μM inhibited Kir2.3(I213L) currents by 47.5±2.6% which was not significantly different from that of Kir2.3 currents (p > 0.05). Kir3.4* has a property of a weak PIP2 interaction but genistein did not have a visible effect on Kir3.4* currents. (5) Chimeras between Kir2.3 and Kir2.1 channels were constructed to identify molecular basis that distinguished the effect of genistein on these channels. It was found that the transmembrane domains and the pore region of Kir2.3 channels were important determinant for high sensitivity for genistein inhibition.Conclusion: Genistein significantly inhibited Kir2.3 currents. The inhibitory effect of genistein on Kir2.3 currents did not involve an alteration of protein tyrosine phosphorylation. Transmembrane domains and pore region of Kir2.3 channels were both important for genistein-induced inhibition of Kir2.3 currents. Understanding the mechanism of genistein action will promote our understanding of structure-function relationship of Kir channels as well as development of potent and specific modulators of Kir channels.SUMMARY1 Kir2.3 channels could be functionally expressed in Xenopus oocytes by microinjecting the transcribed cRNA. The Kir2.3 currents could be observed by using two electrodes voltage clamp technique and the current had the characteristics of inwardly rectification. Ba2+ blocked Kir2.3 currents. PMA, an activator of PKC, concentration-dependently inhibited the currents of Kir2.3 channel. PKC blocker prevented PMA effect. PMA affected Kir2.3 currents by PKC activation.2 Characteristic interactions with PIP2 determined the modulation of Kir2.0 channels by PKC. Kir2.3 channels were not phosphorylated under the condition of PMA treatment. The protein internalization might be involved in PKC inhibition of Kir2.3 currents. The characteristic interactions between Kir channel-PIP2 may also determine the internalization of Kir channels.3 Genistein significantly inhibited Kir2.3 currents. The inhibitory effect of genistein on Kir2.3 currents did not involve an alteration of protein tyrosine phosphorylation. Transmembrane domains and pore region of Kir2.3 channels were both important for genistein-induced inhibition of Kir2.3 currents. Understanding the mechanism of genistein action will promote our understanding of structure-function relationship of Kir channels as well as development of potent and specific modulators of Kir channels.
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