骨質疏鬆症(Osteoporosis)會造成骨頭的脆弱，使患者容易發生骨折。 在正常的狀態下骨骼會不斷進行重塑(remodeling)，並且維持動態平衡，為了維持骨骼的動態平衡，成骨細胞及噬骨細胞的調控作用扮演很重要的角色，如果噬骨細胞的活性太強而失去平衡、噬骨作用(resorption)的速度超過成骨作用的速度很有可能會導致骨質疏鬆。 Ibandronate sodium (Iban)是一種含氮雙磷酸鹽的藥物，目前已經證實Iban會去抑制噬骨細胞的噬骨作用，但是這種藥物對於發生噬骨作用的噬骨細胞上離子通道的作用所知甚少。 因此在本篇研究中我們利用Iban及其他相關化合物來探討藥物和噬骨前驅細胞上離子通道的交互作用。 我們利用細胞膜箝制(patch-clamp)的技術來評估噬骨前驅細胞(RAW 264.7)的電生理特性。 研究結果顯示在此細胞中有中型電導鈣離子活化型鉀離子通道(IKCa channels)的存在，並且根據先前的研究報告指出此離子通道對於許多疾病具有治療的潛力。 我們同時在噬骨前驅細胞上進行聚合酶連鎖反應中也有得到此離子通道的基因片段，並且透過細胞膜箝技術中之完整細胞模式(whole-cell model)的結果得知，Iban會去抑制鉀離子通道電流並且呈現濃度依賴性的關係，並且透過公式換算得到藥物發揮一半抑制效力的濃度(IC50)為28.9 M。 此鉀離子電流會受到IKCa通道的專一抑制劑TRAM-34所抑制，這種抑制現象會隨著加入DC-EBI0(已知IKCa通道刺激劑)而被逆轉。 其次，我們將含有Iban (10 M)的電解溶液注入細胞測量，鉀離子電流仍然會受到抑制，而再加入inomycin (10 M)之後，這種抑制現象就會被逆轉。 另外，噬骨前驅細胞經過雌激素24小時的處理之後，我們發現Iban 對於噬骨細胞的抑制鉀離子電流作用仍然存在。此外，Iban 也不會對向內糾正鉀離子電流(K(IR))有任何的影響。 在細胞連接片模式(cell-attached model)中，我們發現Iban 會去抑制IKCa通道的活性但是並不會改變此通道的電導。 此外，此離子通道的活性是會受到KN-93 (calmodulin 的抑制劑)所抑制，隨後再加入Iban 並不會更進一步的減少此通道的開啟機率。 再由電流箝制(current-clamp recording)的紀錄指出Iban (10 M) 會造成噬骨前驅細胞的膜電位產生去極化，然後再加入DC-EBIO (10 M)之後去極化的現象就會被逆轉回來。 我們參考其他文獻，進行細胞遷移(cell migration)的實驗，結果顯示由細胞內毒素(lipopolysaccharide)所產生的細胞遷徙現象會被Iban抑制，並且此抑制現象會呈現著濃度依賴性的關係。 最後，我們利用Chembio3D 這套軟體分析Iban和鈣離子結合時化學能的變化，結果暗示鈣離子和Iban有強烈的結合能力。 總結本篇的實驗，透過Iban對於噬骨前驅細胞的抑制作用可以對活體中噬骨細胞的功能活性將會有更進一步的了解。

Osteoporosis is a bone disease in which the bone becomes fragile and increases fracture risk. Bone remodeling plays an important role in bone homeostasis and is regulated by activity of osteoblasts and osteoclasts. Hyperactivity of osteoclasts may cause osteoporosis because the rate of bone resorption exceeds bone formation. Bisphosphonate drug Ibandronate (Iban, Boniva®) has shown to reduce skeletal complications through inhibiting osteoclast-mediated bone resorption. The intermediate-conductance Ca2+-activated K+ (IKCa) channels (also known as KCa3.1, SK4, IKCa1, or KCNN4) are encoded by the KCNN4 gene. These channels have single-channel conductance of 20-60 pS and their modulators represent a potentially therapeutic approach to many diseases including osteoporosis. However, how Iban interacts with these channels in osteoclasts largely remains unclear. In this study, we attempt to investigate whether Iban has any effect on ion currents in osteoclast precursor RAW 264.7 cells. In the RT-PCR experiment, the mRNA expression of KCNN4 could be detected in these cells. The result showed that the amplitude of whole-cell K+ currents (IK) was suppressed by Iban in a concentration-dependence manner with IC50 value of 28.9μM. Under 17β-estradiol treatment in RAW cells for 12 hours, Iban-induced inhibition of IK remained effective. Moreover, this compound alone did not affect inwardly rectifying K+ current in RAW cells. IK amplitude was inhibited by TRAM-34, a specific IKCa blocker, and Iban. Iban-mediated inhibition of IK was reversed by DC-EBIO, an opener of (IKCa) channels. Inhibitory effects on IK caused by Iban (10 μM) in the pipette was reversed by bath addition of ionomycin (10 μM). In cell-attached recordings, Iban added to bath did not affect single-channel conductance of IKCa channels. Instead, it did significantly decrease the channel activity. IKCa-channel activity was also suppressed by KN-93, a calmodulin inhibitor. Subsequent addition of Iban did after calmodulin inhibitor did not decrease the channel open probability further. In current-clamp recordings, Iban caused membrane depolarization of RAW cells, and DC-EBIO reversed the Iban-induced depolarization. In cell migration assay, Iban suppressed lipopolysaccharide-induced migration in a concentration-dependent manner. With the aid of ChemBiol3D analysis, as Ca2+ ions are exposed to Iban, the degree of freedom becomes strongly restricted. Our results suggest that the inhibition of IKCa channels caused by Iban could be an important mechanism underlying the activity of osteoclasts occurring in vivo.

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