Bastiaanse E M, Jongsma H J, van der Laarse A, Takens-Kwak B R
Department of Cardiology, University Hospital, Leiden, The Netherlands.
J Membr Biol. 1993 Nov;136(2):135-45. doi: 10.1007/BF02505758.
To assess whether alterations in membrane fluidity of neonatal rat heart cells modulate gap junctional conductance (gj), we compared the effects of 2 mM 1-heptanol and 20 microM 2-(methoxy-ethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)-octanoate (A2C) in a combined fluorescence anisotropy and electrophysiological study. Both substances decreased fluorescence steady-state anisotropy (rss), as assessed with the fluorescent probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) by 9.6 +/- 1.1% (mean +/- SEM, n = 5) and 9.8 +/- 0.6% (n = 5), respectively, i.e., both substances increased bulk membrane fluidity. Double whole-cell voltage-clamp experiments showed that 2 mM heptanol uncoupled cell pairs completely (n = 6), whereas 20 microM A2C, which increased bulk membrane fluidity to the same extent, did not affect coupling at all (n = 5). Since gap junction channels are embedded in relatively cholesterol-rich domains of the membrane, we specifically assessed the fluidity of the cholesterol-rich domains with dehydroergosterol (DHE). Using DHE, heptanol increased rss by 14.9 +/- 3.0% (n = 5), i.e., decreased cholesterol domain fluidity, whereas A2C had no effect on rss (-0.4 +/- 6.7%, n = 5). Following an increase of cellular "cholesterol" content (by loading the cells with DHE), 2 mM heptanol did not uncouple cell pairs completely: gj decreased by 80 +/- 20% (range 41-95%, n = 5). The decrease in gj was most probably due to a decrease in the open probability of the gap junction channels, because the unitary conductances of the channels were not changed nor was the number of channels comprising the gap junction. The sensitivity of nonjunctional membrane channels to heptanol was unaltered in cholesterol-enriched myocytes. These results indicate that the fluidity of cholesterol-rich domains is of importance to gap junctional coupling, and that heptanol decreases gj by decreasing the fluidity of cholesterol-rich domains, rather than by increasing the bulk membrane fluidity.
为了评估新生大鼠心脏细胞的膜流动性改变是否会调节缝隙连接电导（gj），我们在一项结合荧光各向异性和电生理学的研究中比较了2 mM 1-庚醇和20 μM 2-(甲氧基-乙氧基)乙基8-(顺式-2-正辛基环丙基)-辛酸酯（A2C）的作用。用荧光探针1-(4-三甲基铵苯基)-6-苯基-1,3,5-己三烯（TMA-DPH）评估，两种物质均降低了荧光稳态各向异性（rss），1-庚醇使rss降低了9.6±1.1%（平均值±标准误，n = 5），A2C使rss降低了9.8±0.6%（n = 5），即两种物质均增加了整体膜流动性。双全细胞电压钳实验表明，2 mM庚醇使细胞对完全解偶联（n = 6），而20 μM A2C虽然使整体膜流动性增加到相同程度，但对细胞偶联没有任何影响（n = 5）。由于缝隙连接通道嵌入在膜中相对富含胆固醇的区域，我们用脱氢麦角固醇（DHE）专门评估了富含胆固醇区域的流动性。使用DHE时，庚醇使rss增加了14.9±3.0%（n = 5），即降低了胆固醇区域的流动性，而A2C对rss没有影响（-0.4±6.7%，n = 5）。在细胞“胆固醇”含量增加后（通过用DHE加载细胞），2 mM庚醇并未使细胞对完全解偶联：gj降低了80±20%（范围41 - 95%，n = 5）。gj的降低很可能是由于缝隙连接通道开放概率的降低，因为通道的单位电导没有改变，组成缝隙连接的通道数量也没有改变。在富含胆固醇的心肌细胞中，非连接膜通道对庚醇的敏感性未改变。这些结果表明，富含胆固醇区域的流动性对缝隙连接偶联很重要，并且庚醇通过降低富含胆固醇区域的流动性而非增加整体膜流动性来降低gj。
