Functional Roles Of A Ca～(2+)-Activated K～+ Channel In Atrioventricular Node

Background and objectiveThe atrioventricular node(AVN) is a highly specialized pacemaking tissue located at the junction of the right atrium and ventricle. AVN is the only electrical connection between atrium and ventricles and therefore it is responsible for transmitting electrical excitation from the sinoatrial node and atrium to working ventricular muscle. Electrical activity is conducted slowly through the AV node, which results in a delay between atrial and ventricular contraction, ensuriong that ventricular contraction does not begin before atrial contraction has ended. In some disease states, the AV node provides further functions. If the sinoatrial node or atrial conduction fails, the AV node becomes the ventricular pacemaker by its own spontaneous rhythm.Despite this vital roles, the electrophysiology of the AV node is still poorly understood. Studies have identified the roles of several ion channels in the AVN function and recent work has begun to identified an array of ion channel genes in the pacemaking tissues. Small-conductance Ca²⁺-activated K⁺ channels (SK channels) are voltage independent and are activated by intracellular Ca²⁺ ions. There are three SK channel subtypes (SK1-SK3) expressed in mammalian brain. SK channels are encoded by at least three genes: SK1, SK2, and SK3 (KCNN1, KCNN2, and KCNN3), with differential sensitivity toward apamin, a selective SK channel antagonist. SK2 is highly sensitive to apamin, whereas SK1 channels are not affected by apamin. SK3 channels are intermediate.SK channels are widely expressed in different tissues, including the brain, peripheral nervous system, liver and smooth muscle. However, their presence and functional significance have not been well studied in the heart. Dr.Chiamvimonvan's lab has recently identified SK channels in human and mouse cardiac myocytes are highly expressed in atrial compared to ventricular tissues. SK channels are important in the repolarization of cardiac action potentials in mice and humans. Furthermore, using genetically mouse model with over-expression of SK2 channel showed evidences of AVN and atrioventricular(AV) conduction dysfunction.In this paper, we test the functional properties of the AVN utilizing overexpression and knock-out of SK2 channel mice and develop an important information with regards to the functional roles of SK2 channel in AVN cells. Utilizing. HEK293 cells expressed SK2 channel, we investigated the molecular mechanisms of SK2 channel which is associated with a-actinin2, an important binding protein of actin cytoskeleton。Methods and Results1. Over-expressing of SK2 channel(SK2+/T), knock-out of SK2 channel(SK2+/⊿)mice with C57B1/6J background and WT mice(16-22 weeks, both sexes) were used in this study.2. SK2+/T and SK2+/⊿ mice show evidence of SA and AV node dysfunctionSurface ECG recordings showed significant sinus bradycardia with prolongation of the PR intervals in SK2+/â–³mice compared to WT amimals.In contrast, SK2+/T mice showed significant shortening of the RR and PR intervals.These differences in the P-R and R-R intervals may represent abnormalities in the pacemaking activities in SA and AV nodal cells in the gene-targeted mice.3. SK2+/T nice show an increase the firing frequency of A VN while the opposite findings were observed in SK2+/⊿ miceThe mice were anesthetized by intraperitoneal injection of pentobarbital sodium (60mg/kg). The heart was excised and the right atrium was opened. The AV node region was removed. Spontaneous action potentials(APs) were recorded from isolated AV nodal preparations from SK2+/T, SK2+/â–³, WT mice using microelectrode techniques with 3 mol/L KCl microelectrodes at 33℃.APs recorded from the AVN preparation can be identified by the presence of the slow diastolic depolarization and a very slow upstroke of phase 0. SK2+T mice showed a significant increase in the spontaneous activities of the AVN compared to WT controls.In contrast, SK2+/â–³mice showed a significant decrease in the firing frequency of the AVN compared to the WT group. Detailed analysis of the APs reveals significant changes in the CL, APD as well as DDR in the SK2+/T and SK2+/â–³mice. Over-expression of SK2 channel in SK2+/T mice resulted in a significant shortening of APD80, while APD80 was significantly prolonged in SK2+/â–³mice compared to WT animals.Moreover, SK2+/T mice showed a significant increase in the DDR and a corresponding shortening in the CL compared to WY control. The opposite effects were observed in SK2+/â–³mice.4. Single AVN cell Ik, ca recordingSingle AVN cells were isolated from WT, SK2+/T and SK2+/â–³mice as previously described with some modification. Whole-cell Ca²⁺-activated K⁺ current (Ik, ca)was recorded from single AVN cells at room temperature using patch-clamp technique. The current density elicited from a holding potential of-55mV, by the voltage-steps from-90mV to +40mV. Whole -cell current records Were filtered at 2 kHz and sampled at 10kHz. Apamin-semsitive K⁺ current were obtained using digital subtraction(before and after application of 500 pmol/L apamin).The current density-voltage relationships of apamin-sensitive K⁺ current showed the inwardly rectifying currents which is decreased with increasing depolarization voltages. Both the inward and outward components of the Ik,ca were blocked by apamin. The reversal potential was -80mV, which is corresponding with Nernst equation.Over-expressing of SK2 channel in SK2+/T mice resulted in a significant increase in the apamin-sensitive current density in AVN cells and a significant decrease in the current density in the single AVN cells isolated from SK2+/â–³mice compared to WT animals. The results display evidence of the present of Ik, ca in the AVN cells.5. Double immunofluorescence labeling and Con focal MicroscopySingle isolated AVN cells from WT and SK2â–³/â–³mice were fixed with 4% paraformadehyde, then blocked with 1% BSA, incubated overnight at 4℃with primary antibodies. The following primary antibodies were used:(1)anti-SK2 antibody (1:100 dilution)& anti-neurofilamin (NF160 KD)antibody (1:100);(2)anti-SK2 antibody (1:100)& anti-a-actinin2 antibody(1:800).Confocal microscopy was performed by treatment with FITC-conjugated goat anti-rabbit antibody(1:250)and TEXRED-conjugated goat anti-mouse antibody(1:250). Control experiments were performed by secondary antibody only under the same experimental conditions.Confocal microscopy illustrated the positive SK2 staining in single AVN cell as well as atrial and ventricular myocytes. NF160, a marker of the pacemaker and conduction system, was only expressed in AVN cell. The absence of positive SK2 staining was detected in the AVN cells in the SK2â–³/â–³mice.6. Expression of SK2 protein in WT mice AVNThe sections of the hearts from the WT and SK2â–³/â–³mice were blocked in 5% goat serum,then treated with anti-SK2 antibody(1:200). The sections were then treated with biotinylated secondary antibody.Expression of SK2 protein in the AVN and working myocardium in WT animals was documented as brown positive staining and the absence of SK2 protein expression in the AVN in the SK2â–³/â–³mice was detected. AVN region was further evaluated in cardiac sections stained with H&E and Trichrome.7. HEK293 cell culture and plasmids transfectionHEK293 cells were used as a transient expression system. The cells were maintained in Dulbecco's modified Eagle's medium at 37℃in an air/5%CO₂ incubator. HEK293 cells were transfected using the following plasmids: 1) pSK2-IRES-EGFP & pcDNA3-a-actinin 2;2)pSK2-IRES-EGFP using Lipofectamine TM 2000 according to the manufacturer's protocol. Whole-cell Ik, ca recorded from HEK 293 cells was performed at room temperature 36-48 hrs after transfection using the same recording protocol as for the AVN cells. The cells coexpressing SK2 channel plusα-actinin2 were pretreated with 2.5 pmol/L Cytochalasin D for 4 hrs before the recording.8. Modulation of Ik, ca by a-actinin 2 in HEK 293 cellsIk, ca density from HEK 293 cells expressedα-actinin 2 and SK2 channel was significantly larger than the current density from HEK 293 cells exresssed SK2 channel alone.Ik, ca density increase-4 fold when SK2 channel was co-expressed with a-actinin2 compared to SK2 channel expressed alone. No apamin-sensitive current was measured in non-transfected HEK 293 cells. Pre-treatment with cytochalasin D significantly reduced Ik, ca current density in HEK 293 cells expressed bothα-actinin2 and SK2 channel.9. Expession and subcellular co-localization of SK2 channel and a-actinin2 in HEK293 cellsFor transfected cells, immunostaining was performed 2-3 days post-transfection. The cells were fixed with 2% paraformadehyde, then blocked with 1% BSA, incubated overnight at 4℃with primary antibodies. The following primary antibodies were used:(1)anti-SK2 antibody (1:100 dilution)+anti-a-actinin2 antibody (1:800).Confoeal microscopy was performed by treatment with FITC-conjugated goat anti-rabbit antibody(1:250)and TEXRED-conjugated goat anti-mouse antibody (1:250). Control experiments were performed by secondary antibody only.Western blot documented the presence of a 98 kDa protein as expected forα-actinin2 in HEK 293 cells transfected withα-actinin2 and SK2 plasmids. The SK2 antibody recognizes a 60-kDa SK2 monomer and a higher molecular weight form, 120 kDa dimer.Confocal microscopy showed that SK2 channel andα-actinin 2 were co-localized in the plasma membrane of HEK 293 cells co-transfected with both SK2 andα-actinin 2 plasmids, suggesting thatα-actinin2 interacted with SK2 channel. Conclusion and DiscussionAlthough a few studies have described the electrophysiology of rabbit AVN, very little is known about the electrophysiological property of small animals AVN, including mouse AVN. The AVN is located at the junction of the right atrum and ventricular septum. There are fiber tissues around the compact node, which makes single cell isolation more difficult. Moreover, the small size of the AVN in mouse limites the study of single AVN cells using the electrophysiology, immunofluorescence, Western blot and so on. In this study, we describe an isolation procedure of mouse single AVN cells. Thus it offers considerable potential for the study of the gene-targeted mouse models including those which display abnormal pacemaker function.The two gene-targeted mouse models used in our study with over-expression and knock-our of SK2 channel allow us to directly test the functional roles of SK2 channel in the whole animal, AVN preparations as well as single isolated AVN cells. Overexpression of SK2 channels resulted in the increase in the repolarization of the AVN cells associated with an increase in the spontaneous activity. On the other hand, SK2 channel knock-out resulted in the opposite effects on the AVN activity. Detailed analysis of the spontaneous AP revealed significant changes in the APD and DDR in the SK2+/T and SK2+/â–³mice compared to WT control. The increasing firing frequency of the SK2+/T mice AVN is related to a significant shortening of APD80 and significant increasing in the DDR caused by over-expression of SK2 channel. The opposite effects were observed in the SK2 knock-out mice. The slow spontaneous activity of SK2+/â–³mice AVN is associated with a significant prolonged APD 80 and decrease DDR.To further document that the changes in the AVN observed in the SK2+/T and SK2+/â–³mice were indeed due to differing the expression of SK2 channel, we directly detected the Ik, ca measured as apamin-sensitive K⁺ current density in single isolated AVN cells from SK2 transgenic and knock-out animals using the same recording protocal as for the atrium myocytes. Whole-cell Ik, ca was recorded using voltage steps from a holding potential of-55 mV to inactivate the transient outward K⁺ currents, which were known to be present in mouse AVN myocytes. Na⁺ and Ca²⁺ ions were eliminated from the external solution. The interior of the cells was dialyzed using pipette solutions containing known concentrations of Ca²⁺. Using the above recording condition, a time-independent inward rectifier K⁺ current could be recorded. In addition, the component of both inward and outward current could be blocked by application of apamin. The results showed a significant increase in the apaminsensitive current density in AVN cells isolated from SK2+/T and a significant decrease in the current density in the SK2+/â–³mice compared to WT controls. The prolongation of ADP80 in the AVN in SK2 knock-out mice is due to downregulation of Ik, ca in the AVN, wherase the shortening of APD80 in SK2+/T mice upregulation of Ik, ca in the AVN.To determine whether the dysfunction of AVN in over-expression and knock-out SK2 channel is consistent with the expression of the SK2 channel, we detected the expression of SK2 subunite in heart AVN section and single AVN cells using immunohistochemistry and confocal. The expression of SK2 in AVN cells was localized to the surface membrane, whereas the immunofluorescence signal of SK2 channel in the atria and ventricular myocetes was localized to T-tubules which specialized regions of surface membrane which extend into the cell interior associated with the excitation-contraction of cardiac working myocytes.Actinin is members of a family of actin-binding cytoskeletal protein that may link the cytoskeleton to membrane proteins such as ion channel. Previous work has demonstrated that ion channels undergo highly specialized subcellular localization at the plasma membrane which is crucial to cell function. Muscle actinin isoformα-actinin2 binds and modulates the functions of a voltage-gated K⁺ channel, Kv1.5, NMDA-type glutamate receptor, L-type Ca²⁺ channel(Cav1.2)and so on.To seek molecular that bind toα-subunit of the SK2 channel, yeast two-hybrid system revealed the interaction of SK2 channel c-terminus withα-actinin2. In vitro biochemical methods and confocal were applied to confirm the interactions betweenα-actinin2 protein and SK2 channel. Our data showed the functional significance of the interaction betweenα-actinin2 and SK2 channel. The Ik, ca density from HEK 293 cells transfected with bothα-actinin2 and SK2 channel was significantly large than current density from HEK 293 cells transfected with SK2 channel alone. Furthermore, L-type Ca²⁺ channels were found to be co-localized with a-actinin2 cytoskeletal protein. Knock-out Cav1.3 channel in the heart resulted in downregulation of SK2 channel in the mouse cardiac myocytes. The functions of SK2 channels in the heart are dependent on normal expression of Cav1.3 channel in mouse atrial myocytes. The co-localization of SK2 channel with Cav1.3 channel may be associated with theα-actinin2 cytoskeletal protein.α-actinin2 modulates the SK2 channel by different molecular mechanisms. One possible molecular is Calmodulin(CaM). Bothα-actinin2 and SK2 C-terminus contain a CaM binding domain. Moreover, CaM is a Ca²⁺ sensor for all of Ca²⁺-activated K⁺ channels. Indeed, one study has demonstrated that CaM is known to compete withα-actinin2 for binding to another ion channel, the NMDA receptor, thereby mediating both inactivation of the channel and their localization.Evidence indicated that Cytochalasin D accelerated cardiac ATP-sensitive K⁺ channel rundown and directly influence the interaction between actin and actinin. Indeed, we found that Cytochalasin D modulated SK2 channel function in the transfected HEK293 cells. Cytoskeletal networks may exert an interaction with SK2 protein, directly and indirectly modulate its channel function, in addition to help its anchorage to the membrane. This study points the way to new regulatory mechanisms for cardiac ion channel.SK channels have been shown to play an important role in setting the tonic firing frequency of neurons. Their activation causes membrane hyperpolarization, which inhibits cell firing and limits the frequency of repetitive APs. The increase in intracellular Ca²⁺ evoked by AP firing allows SK channels activation to generate a long lasting hyperpolarization. This hyperpolarization protects the cell from the deleterious effects of continuous tetanic activity. In contrast to the hyperpolarization effects of SK channels in neuron, in cardiac myocytes, the SK channels contributed markedly toward the late phase of the cardiac repolarization which is susceptible to extra excitation, e.g. early after depolarization and arrhythmias. Here, we demonstrate that over-expression of SK2 channel in the heart increases the firing frequency of the pacemaking tissues. Therefore, the SK2 channel may serve a distinct role to potentiate the increase in AVN conduction during exercise or sympathetic responses under normal physiological condition. On the other hand, an increasing intracelluar Ca²⁺ under pathologic conditions may produce profound changes in AVN conduction. For example, during atrial fibrillation, the rapid depolarization may increase intracellular Ca²⁺ and potentiate the Ik, ca and AVN conduction. Hence, SK2 channel may modulate atrioventricular conduction during atrial arrhythmias.Take together, here we first documented the AVN dysfunction in the SK2+/T and SK2+/â–³mice. Over-expression of SK2 channel in the heart increased the frenquency of firing of the AVN. Knock-out of SK2 channel in the heart caused bradycardia and delays atrioventricular (AV)conduction. Furthermore, over-expression of SK2 channel in the heart increased the rates of diastolic depolarization and repolarization due to upregulation of Ikca in the AVN. Knock-out of SK2 channel in the heart slowed the rates of diastolic depolarization and prolonged the duration of repolarization because of downregulation of Ikca.In addition, in the present study, we first employed confocal immunohistochemical and eletrophysiological techniques to demonstrate that the association of a-actinin2 with SK2 channel localizes the channel to the membrane and regulates the channel function. Our data present the first study on the molecular mechanism of coupling of SK2 channel with cytoskeletal proteins. 论文部分小电导-Ca(2+)-激活K⁺通道在小鼠房室结细胞的表达及其功能的研究#1引言#1第一部分SK2通道在小鼠房室结细胞的功能性作用#31．前言#32．材料和方法#33．实验结果#84．讨论#17参考文献#22第二部分α-actinin2对SK2通道的功能性调节#271．前言#272．实验材料和方法#283．实验结果#324．讨论#36参考文献#38综述部分小电导-Ca²⁺-激活K⁺通道#41参考文献#54后记缩略词表#66读博期间发表的论文#68致谢#69...