Liu G H, Oba T
Department of Physiology, Nagoya City University Medical School, Japan.
Jpn J Physiol. 1990;40(5):723-36. doi: 10.2170/jjphysiol.40.723.
We studied the relationship between surface charge and release of Ca2+ in the heavy sarcoplasmic reticulum (SR) of skeletal muscle. The inner and outer surface potentials and charge densities of the membrane treated with a lipophilic anion, tetraphenylboron (TPB-), were measured using 1-anilino-8-naphthalene-sulfonate fluorescence. Ca2+ was loaded passively or actively by the SR. Ca2+ release was estimated by the fluorescence of chlortetracycline, and protein conformational change was monitored by use of the sulfhydryl group fluorescent probe, N-(7-dimethylamino-4-methyl-3-coumarinyl) maleimide (DACM). Treatment of Ca2(+)-loaded SR vesicles with micromolar TPB- dose-dependently increased the local fixed negative charge on the inner surface, and changed the DACM fluorescence intensity in parallel with the Ca2+ release. The changes in surface charge and in DACM fluorescence intensity did not originate from the Ca2+ flux. A lipophilic cation, tetraphenylarsonium (TPA+), screened the negative inner surface charge which was increased by TPB-, and inhibited both TPB(-)-induced change in DACM fluorescence intensity and Ca2+ release. Millimolar Mg2+ reduced degrees of TPB(-)-induced Ca2+ release from the SR and of TPB(-)-induced contraction in mechanically skinned fibers. Mg2+ did not inhibit the increase in the negative inner surface charge and DACM fluorescence intensity produced by TPB-. Thus, the local increase in negative charge on the SR inner membrane leaflet seems to be causally related to the Ca2+ release. Mg2+ and TPA+ are suggested to inhibit TPB(-)-induced Ca2+ release by different mechanisms.
我们研究了骨骼肌重肌浆网（SR）中表面电荷与Ca2+释放之间的关系。使用1-苯胺基-8-萘磺酸盐荧光法测量了用亲脂性阴离子四苯基硼（TPB-）处理的膜的内外表面电位和电荷密度。Ca2+通过SR被动或主动加载。通过金霉素的荧光估计Ca2+释放，并使用巯基荧光探针N-（7-二甲基氨基-4-甲基-3-香豆素基）马来酰亚胺（DACM）监测蛋白质构象变化。用微摩尔浓度的TPB-处理加载了Ca2+的SR囊泡，可剂量依赖性地增加内表面的局部固定负电荷，并与Ca2+释放平行地改变DACM荧光强度。表面电荷和DACM荧光强度的变化并非源于Ca2+通量。亲脂性阳离子四苯基砷（TPA+）屏蔽了由TPB-增加的内表面负电荷，并抑制了TPB-诱导的DACM荧光强度变化和Ca2+释放。毫摩尔浓度的Mg2+降低了TPB-诱导的SR中Ca2+释放程度以及TPB-诱导的机械去表皮纤维收缩程度。Mg2+并未抑制TPB-产生的内表面负电荷增加和DACM荧光强度增加。因此，SR内膜小叶上负电荷的局部增加似乎与Ca2+释放存在因果关系。Mg2+和TPA+被认为通过不同机制抑制TPB-诱导的Ca2+释放。
