Ibandronate Sodium a Nitrogen-Containing Bisphosphonate Inhibits Intermediate-Conductance Calcium-Activated Potassium Channels in RAW264 7 Cells

摘要

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

獎項日期	2015 1月 14
原文	English
監督員	Sheng-Nan Wu (Supervisor)