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2010 생명의약연구원 월례정기세미나 세미나 주제 “이온통로와 질환” 세미나 일정 2010년 12월 15일 17:00~ 19:30 (수요일) 세미나 장소 중앙대학교 약대 교수 세미나실 연자; (20분 발표, 10분 질문) 1. 강원대학교 의과대학 생리학교실, 박원선 교수(17:10~17:40) 2. 중앙대학교 의과대학 생리학교실, 고재홍 교수(17:40~18:10) 3. 성균관대학교 의과대학 생리학교실, 강동묵 교수(18:10~18:40) 4. 서울대학교 의과대학 생리학교실, 김성준 교수(18:40~19:10) 박원선교수; Patho-, Physiological role of inward rectifer K+ channels in small diameter coronary arteries K+ channels play indispensable roles in establishing the membrane potential and in regulating the contractile tone of arterial smooth muscle cells. There are four types of K+ channels in arterial smooth muscle: voltage-dependent K+ (KV), Ca2+-dependent K+ (BKCa), ATP-dependent K+ (KATP), and inward rectifier K+ (Kir2) channels. Comparatively few physiological studies have focused on Kir2 channels, because they are present only in certain small-diameter cerebral and submucosal arterioles, as well as in coronary arterial smooth muscle. Here, we review the characteristics and regulation of Kir2 channels in vascular arterial smooth muscle. Current knowledge of the predominant Kir2 channel subtype is Kir2.1, not Kir2.2 and 2.3. Electrophysiological measurements to determine the current-voltage relationship in arterial smooth muscle revealed inward rectification with a single-channel conductance of 21 pS. Kir2 channels were found to influence the resting tone of cerebral and coronary arteries based on the fact that barium (Ba 2+) induces the constriction of these arteries at resting tone. Kir2 channels are also highly responsive to vasoconstrictors and vasodilators. For example, the vasoconstrictors endothelin-1 and angiotensin II inhibit Kir2 channel function by activating protein kinase C (PKC), and the vasodilator adenosine stimulates Kir2 channel function by increasing the level of cAMP, which subsequently activates protein kinase A (PKA). Certain pathological conditions such as left ventricular hypertrophy are associated with a decrease in Kir2 channels expression. Although our understanding of the physiological role and regulation of Kir2 channels is incomplete, it is believed that Kir2 channels contribute to the control of vascular tone in small-diameter vessels via various intracellular signalling pathways that regulate cell membrane potential. Key Words: inward rectifier K+ channel, protein kinase C, protein kinase A, vasoconstrictor, vasodilator, hypertrophy 고재홍 교수 Cloning of large-conductance Ca2+-activated K+ channel alpha-subunits in mouse cardiomyocytes Large-conductance Ca2+-activated K+ (BKCa) channels are widely distributed in cellular membranes of various tissues, but have not previously been found in cardiomyocytes. In this study, we cloned a gene encoding the mouse cardiac BKCa channel α-subunit (mCardBKa). Sequence analysis of the cDNA revealed an open reading frame encoding 1154 amino acids. Another cDNA variant, identical in amino acid sequence, was also identified by sequence analysis. The nucleotide sequences of the two mCardBKa cDNAs, type 1 (mCardBKa1) and type 2 (mCardBKa2), differed by three nucleotide insertions and one nucleotide substitution in the N-terminal sequence. The amino acid sequence demonstrated that mCardBKa was a unique BKCa channel α-subunit in mouse cardiomyocytes, with amino acids 41-1153 being identical to mouse Slo1 and amino acids 1-40 corresponding to Kcnma1. These findings suggest that a unique BKCa channel α-subunit is expressed in mouse cardiomyocytes. 강동묵 교수 Regulation of skeletal muscle differentiation by store-operated Ca2+ channels Store-operated Ca2+ entry (SOC) channels are known to activate by the depletion of calcium inside of endoplasmic reticulum (ER) of the cells. By the activation of SOC channels, entry of extracellular calcium into the cytoplasm replenishes ER Ca2+ stores. Recently, stromal interaction molecule (STIM1, STIM2) and the Orai family of plasma membrane channels (Orai1, Orai2, Orai3) have been discovered as the molecular identity of SOC channels. It is now well established that STIM is the Ca2+ sensor protein on ER membrane and is required to activate SOC channels. Upon depletion of ER calcium store, STIM1 redistribute to plasma membrane and binds with the Orai1 Ca2+ channel to form SOC channels. It has been recognized that STIM1 and Orai1 are highly expressed in skeletal muscle cells, and genetic ablation of STIM1 or Orai1 produces a severe growth defect of mice. In the present study, we used genetic ablation or over-expression of STIM1 to investigate the role of STIM1 during the differentiation process of C2C12 skeletal muscle cell line. In addition, the functional interaction of STIM1 with a Ca 2+dependent phosphatase (calcineurin) and a transcriptional factor (NFATc) was investigated. STIM1, STIM2 and Orai1 gene expression is markedly increased by the induction of C2C12 skeletal muscle differentiation. As a consequence, the activity of SOC was higher in differentiated myotubes than proliferating myoblasts. A commonly used SOC inhibitor, 2-APB, markedly inhibited the muscle differentiation. Over-expression of STIM1 enhanced the SOC activity and the myoblast differentiation, whereas knock-down of STIM1 using siRNA technique produced apposite effects. When the myoblasts were treated with calcineurin inhibitors (cyclosporine A or FK506), expression of STIM1, STIM2, the SOC activity, and the degree of myoblast differentiation were all decreased. Luciferase assay revealed that the ablation of STIM1 decreased the activity of NFATc, which is a transcription factor dictated by activated calcineurin, whereas over-expression of STIM1 exert opposite effect on NFATc activity. As expected, calcineurin inhibitors markedly decreased NFATc activity. Taken together, we suggest that STIM1-calcineurin-NFATc signaling pathway is important for regulating skeletal muscle differentiation processes. 김성준 교수 Exercise training increases K+ channel currents and augments K+-mediated vasodilation in deep femoral artery of rats Aims: A moderate increase in extracellular [K+] ([K+]e) induces relaxation of small arteries via activating inwardly rectifying K+ current (IKir). The K+-vasodilation is an important mechanism for exercise-induced hyperemia in skeletal muscle. We investigated whether IKir and K+-vasodilation are enhanced in deep femoral arteries (DFA) from exercise-trained rats (ET-rats, treadmill-running (20 m/min, 20 min, six days for two week). The effects of ET on K+-vasodilations and IKir were also compared with cerebral arteries and mesenteric arteries. Methods and Results: The K+-vasodilation of DFA and the density of IKir and voltagegated K+ current (IKv) were increased in ET-rats. The myogenic tone of DFA was unchanged in ET-rats. Although the functional up-regulations of IKir and IKv were also observed in cerebral arteries, the K+-vasodilation was not increased in ET-rats. Interestingly, background Na+ conductance was also increased in the cerebral arterial myocytes while not in DFA myocytes from ET-rats. Neither IKir nor K+-vasodilation was observed in the mesenteric arteries of ET-rats. Conclusion: We firstly report that regular exercise up-regulates IKir in the myocytes of DFA and cerebral artery. Albeit the common increase of IKir, augmentation of K+relaxation was observed in DFA only, which might be due to the increased Na+ conductance in cerebral artery of ET-rats. The increases of IKir and K+-vasodilation of skeletal arteries suggest novel mechanisms of improved exercise hyperemia by physical training. Keywords: exercise training, skeletal artery, smooth muscle, inward rectifying K+ channel