Role Of Lipid Raft In Specificity Of Cell Signaling Modulation Of M Potassium Currents

M current is wildly distributed potassium current in neuronal system, and is one of the major mechanisms controlling the neuronal excitability. M current is the only current activated around threshold potential of neurons and its functional depression could lead to the increased excitability of the neurons, leading to the diseases such as epilepsy. It is known that many neuronal transmitters and peptides can modulate the M currents such the excitability of neurons. Recently, great achievements have been made on the understanding of molecular mechanism for M current modulation, represented by the mechanisms underling the M current modulation by two G protein coupled receptor pathways, M type acetylcholine receptor (M₁) and type II bradykinin receptor (B₂). Although activation of both receptors can induce M current inhibition, the underlying mechanisms are different, with key difference in the effects on intracellular Ca²⁺ concentration. However, it is still an open question as to what cause this difference. This study is intended to understand the mechanism of M current modulation by different cell signaling pathways from the point of lipid raft. Lipid raft is a microdomain structure on the cell membrane, with sizeranging from 55-300 nm. Lipid raft is enriched with cholesterol and sphingomyelin and characterized by low buoyancy density and detergent insolubility. Recent studies have reported that lipid rafts play important roles in cell signaling transduction; the signaling molecules concentrated in the lipid raft might determine the specificity of various cell signaling. This research will focus on the distribution of M1 receptor, B₂ receptor and signaling molecules related to these receptors in lipid rafts and uncover the role of lipid raft in M potassium channel modulation. Objective: To identify the role of lipid raft in specificity of M channel modulation by cell signaling.Methods:1 Isolation and identification of lipid rafts and signaling molecules1.1 Isolation of lipid rafts through sucrose density gradient centrifugationSuperior cervical ganglion (SCG) were isolated from forty 7-day SD rats and lysed with a glass homogenizer. Homogenate was spun at 5,000 rpm for 5 min at 4℃. 1.5 ml of the supernatant was placed in ultracentrifuge tubes on ice and on top of that, 1.5 ml of 80% sucrose was placed and mixed with gentle pipetting to prevent the formation of bubbles. To this, 3.0 ml of 35% sucrose was overlaid, followed by 3.0 ml 5% sucrose. The tubes were then centrifuged at 39,000 rpm for 21 h at 4℃. After centrifugation, nine sequential fractions of the sucrose gradient 1 ml each were gently removed from the top of the tube and individually aliquoted. 4μl of each fraction was dotted on nylon membrane for dot blots to be detected the distribution of GM1 in each fraction, which is a marker of lipid rafts.1.2 Isolation of lipid rafts through detergentSCG were isolated from ten 7-day SD rats and lysed with a glass homogenizer. Homogenate was spun at 5,000 rpm for 10 min at 4℃. The supernatant was then centrifuged at 40,200 rpm for 1 h at 4℃. After centrifugation, the pellet was re-suspended with 200μl buffer containing 500 mM Na2CO3 and 1% triton, sonicated, sit on ice for 30 min and then spun at 15, 000 g for 30 min at 4℃. After that, detergent soluble and insoluble fractions were separated and denatured with 5×SDS-PAGE sample buffer.1.3 Identification of M1 and B₂ receptors and related signaling molecules in lipid raftProteins were mixed with sample buffer and separated on 12% SDS-PAGE. The separated proteins were transferred at 30 V to NC membranes overnight at 4℃. After blocking the membrane with 5% non-fat milk, the blots were incubated overnight with primary antibodies at 4℃. Antibody dilutions were as follows: caveolin-1(1:1000), B₂R(1:1000), M₁(1:200),Gq(1:200),G11(1:200),PLCβ4 (1:200),PLCβ1(1:200). The membranes were washed for 30 min in TBST, then incubated with secondary antibodies labeled with fluorescence for 3⁴ hrs at room temperature.2 M current recordingIsolated SCG neurons were plant on cover slips. M currents were recorded from SCG neurons through perforated patch clamp technique. The cell membrane was held at -20 mV, and then hyperpolarized to -60 mV. The tail current at -60 mV were recorded. The effects of MβCD treatment on OXO-M and BK-induced M current inhibition were studied.3 Intracellular Ca²⁺ measurementThe SCG neurons were incubated with Flu-4-AM(2.5 nmol/L)for 30 min. F127(0.02%) was used for membrane penetration. After incubation, the cells were washed three times with 0.01 M PBS. The BK-induced intracellular Ca2+ rise was observed before and after application of MβCD using Laser Scanning Confocal Microscope.4 Immunocytochemistry and co-immunoprecipitation4.1 Immunocytochemistry of cavolin-1, M₁R and B₂RSCG neurons cells were fixed with 4% polyoxymethylene, and then were placed in PBS containing 0.2% Triton-100 for 30 min. Special primary antibodies (cavolin-1, M₁R and B₂R ) were added overnight. Fluorescence isothiocyanate secondary or tertiary antibodies were used. The cells were then scanned using the laser scanning confocal microscopy to detect the colocalization of cavolin-1 and M₁R, the colocalization of cavolin-1 and B₂R.4.2 Co-immunoprecipitation of IP₃R and B₂RSCG were isolated from 7-day SD rats and lysed with a glass homogenizer. Homogenate was spun at 5,000 rpm for 10 min at 4℃. The supernatant was then added with 2μl IP3 antibody and rotated at 60 rpm at 4℃overnight. Protein G beads were added the next day and continually rotated at 60 rpm at 4℃for 4 h. The beads were washed and proteins were resolved by SDS-PAGE and transferred to NC membrane. 5% non fat milk was performed to block the membrane. B₂ receptor antibody and fluorescence-labeled secondary antibody were used. All washing steps were performed using TBST. Blots were then scanned using the Odyssey Infrared Imaging System.Results:1 The lipid raft seemed to be present in rat SCG neurons. The sucrose density gradient yielded the best resolution of caveolin-1 in raft fractions. Dense staining for caveolin-1 was present in fractions 3-5 with the highest levels in fraction 4. This suggests that fractions 3 and 4 are the raft fractions. GM₁, another marker of the lipid raft, was also found in fractions 1-5 with the highest levels in fraction 1-3.2 B₂R, Gq, G11, PLCβ₁₄ and PLCβ₁₁ were found in raft fraction. As for M₁ receptor, there was little localization in lipid raft fractions.3 The distribution of caveolin-1, B₂R,M₁R,PLCβ₁₁,PLCβ₁₄, Gq, and G11 were detected in detergent insoluble fraction. However, the proportion of B₂R in insoluble fraction is about 83.9% which is much higher than that of M₁R in insoluble fraction of 14.7%. The proportion of other related proteins in insoluble fraction are: PLCβ₁₄ 19.7%, PLCβ₁₁ 18.6%, Gq 34% and G11 21.6%. After treatment with Mβ₁CD for 1 h at 37℃, the proportion of B₂R in insoluble fraction was significantly reduced by 70%.4 OXO-M induced a 82±6.3% inhibition of M current in SCG neurons before Mβ₁CD treatment; after treatment with Mβ₁CD 10 mM and 5 mM, OXO-M induced a 49±8.7%(significantly smaller than the inhibition before Mβ₁CD treatment, P<0.01)and a 71±4.9%(not significantly different from the inhibition before Mβ₁CD treatment, P>0.05)inhibition of M currents, respectively. BK induced a 77±7.1% inhibition of M current in SCG neurons before Mβ₁CD treatment; after treatment with Mβ₁CD 10 mM and 5 mM, OXO-M induced a 32±8.5%(significantly smaller than the inhibition before Mβ₁CD treatment, P<0.01)and a 37±8.0%(significantly smaller than the inhibition before Mβ₁CD treatment, P<0.01)inhibition of M currents, respectively. These results indicate that BK-induced inhibition of M currents is more sensitive to Mβ₁CD treatment. 5 5 mM Mβ₁CD significantly affected the BK-induced intracellular Ca²⁺ rising in SCG neurons. Thus before and after the Mβ₁CD treatment, BK-induced intracellular Ca²⁺ rising of 128.3±3.1% and 107.4±3.0%(P<0.01 ), respectively.6 In SCG neurons, caveolin-1 seems to have more co-localization with B2R than with M1R.Conclusions:1 Lipid rafts exist in rat SCG tissue cells. B2 receptor and related signaling molecules including G_q, G₁₁, PLCβ₁4 and PLCβ₁ are found in lipid raft fractions; M1 receptor is not found in the raft.2 Lipid rafts play an important role in BK mediated inhibition of M currents in SCG neurons.3 An intact lipid raft is necessary for BK-induced Ca²⁺ rising in SCG neurons.4 B2 receptor is colocalized with caveolin-1 in SCG neurons...