Muñoz A, García L, Guerrero-Hernández A
Departamento de Bioquímica, CINVESTAV-IPN, México D. F. 07000, México.
Biophys J. 1998 Oct;75(4):1774-82. doi: 10.1016/S0006-3495(98)77619-2.
The Ca2+ sensitivity of large conductance Ca2+- and voltage-activated K+ channels (BKV,Ca) has been determined in situ in freshly isolated myocytes from the guinea pig urinary bladder. In this study, in situ denotes that BKV,Ca channel activity was recorded without removing the channels from the cell. By combining patch clamp recording in the cell-attached configuration and microfluorometry of fura-2, we were able to correlate BKV,Ca channel activity with changes in cytoplasmic intracellular [Ca2+] ([Ca2+]i). The latter were induced by ionomycin, an electroneutral Ca2+ ionophore. At 0 mV, the Hill coefficient (nH) and the [Ca2+]i to attain half of the maximal BKV,Ca channel activity (Ca50) were 8 and 1 microM, respectively. The data suggest that this large Hill number was not a consequence of the difference between the near-membrane [Ca2+] ([Ca2+]s) and the bulk [Ca2+]i, indicated by fura-2. High Hill numbers in the activation by Ca2+ of BKV,Ca channels have been seen by different groups (e.g., filled squares in Fig. 4 of Silberberg, S. D., A. Lagrutta, J. P. Adelman, and K. L. Magleby. 1996. Biophys. J. 70:2640-2651). However, such high nH has always been considered a peculiarity rather than the rule. This work shows that a high Ca2+ cooperativity is the normal situation for BKV,Ca channels in myocytes from guinea pig urinary bladder. Furthermore, the Ca50 did not display any significant variation among different channels or cells. It was also evident that BKV,Ca channel activity could decrease in elevated [Ca2+]i, either partially or completely. This work implies that the complete activation of BKV,Ca channels occurs with a smaller increment in [Ca2+]s than previously expected from in vitro characterization of the Ca2+ sensitivity of these channels. Additionally, it appears that the activity of BKV,Ca channels in situ does not strictly follow changes in near-membrane [Ca2+].
在豚鼠膀胱新鲜分离的肌细胞中，已原位测定了大电导钙激活钾通道（BKV,Ca）对Ca2+的敏感性。在本研究中，原位是指在不将通道从细胞中移除的情况下记录BKV,Ca通道活性。通过结合细胞贴附式膜片钳记录和fura-2微荧光测定法，我们能够将BKV,Ca通道活性与细胞质内细胞内[Ca2+]（[Ca2+]i）的变化相关联。后者由离子霉素（一种电中性Ca2+离子载体）诱导产生。在0 mV时，希尔系数（nH）和达到最大BKV,Ca通道活性一半时的[Ca2+]i（Ca50）分别为8和1 microM。数据表明，这个大的希尔数并非fura-2所示的近膜[Ca2+]（[Ca2+]s）与整体[Ca2+]i之间差异的结果。不同研究小组在BKV,Ca通道Ca2+激活过程中都观察到了高希尔数（例如，Silberberg, S. D., A. Lagrutta, J. P. Adelman, and K. L. Magleby. 1996. Biophys. J. 70:2640 - 2651中图4的实心方块）。然而，这种高nH一直被视为一种特殊情况而非普遍规律。这项工作表明，高Ca2+协同性是豚鼠膀胱肌细胞中BKV,Ca通道的正常情况。此外，Ca50在不同通道或细胞之间未显示出任何显著变化。同样明显的是，BKV,Ca通道活性在升高的[Ca2+]i中可能部分或完全降低。这项工作意味着，BKV,Ca通道的完全激活发生时，[Ca2+]s的增量比这些通道Ca2+敏感性的体外特征所预期的要小。此外，似乎原位BKV,Ca通道的活性并不严格遵循近膜[Ca +]的变化。
