Inhibition of annexin V-dependent Ca 2+ movement in large unilamellar vesicles by K201, a new 1,4-benzothiazepine derivative

Abstract

Examination was made of the effect of annexin V on Ca 2+ movement into large unilamellar vesicles (LUV) using fura-2, a calcium-sensitive fluorescent dye. To avoid the possible difficulties relating to the addition of annexin V and/or Ca 2+ in fura-2-loaded LUV, the burst method was used. LUV, preincubated with rat annexin V in the presence of Ca 2+, were collected by centrifugation and resuspended, and then burst with Triton X-100 in the presence of fura-2. Inward Ca 2+ movement across the artificial lipid membrane was measured by determination of fura-2 fluorescence due to the leaked Ca 2+ from ruptured LUV. The observed Ca 2+ signal increased dependent on annexin V and Ca 2+ concentrations, whereas bovine serum albumin did not affect this signal up to 1 μM. Thus, annexin V shows Ca 2+ channel activity in LUV. K201, a novel 1,4-benzothiazepine, inhibited inward Ca 2+ movement into LUV caused by annexin V in a dose-dependent manner. In the presence of 50 nM annexin V and 400 μM Ca 2+, 3 μM K201 showed significant inhibition of Ca 2+ movement due to annexin V, and 50% inhibition was achieved at 25μM K201. On the other hand, diltiazem had no such effect even at 30 μM. K201 is thus shown to have inhibitory activity on inward Ca 2+ movement due to annexin V in artificial vesicles and may prove useful as a probe for elucidating the functions of annexin V in vivo.