In Situ Characterization of the Ca 2+ Sensitivity of Large Conductance Ca 2+-Activated K + Channels: Implications for Their Use as Near-Membrane Ca 2+ Indicators in Smooth Muscle Cells

Abstract

The Ca 2+ sensitivity of large conductance Ca 2+- and voltage-activated K + channels (BK V,Ca) has been determined in situ in freshly isolated myocytes from the guinea pig urinary bladder. In this study, in situ denotes that BK V,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 BK V,Ca channel activity with changes in cytoplasmic intracellular [Ca 2+] ([Ca 2+] i). The latter were induced by ionomycin, an electroneutral Ca 2+ ionophore. At 0 mV, the Hill coefficient ( n H) and the [Ca 2+] i to attain half of the maximal BK V,Ca channel activity (Ca 50) were 8 and 1 μM, respectively. The data suggest that this large Hill number was not a consequence of the difference between the near-membrane [Ca 2+] ([Ca 2+] s) and the bulk [Ca 2+] i, indicated by fura-2. High Hill numbers in the activation by Ca 2+ of BK V,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 n H has always been considered a peculiarity rather than the rule. This work shows that a high Ca 2+ cooperativity is the normal situation for BK V,Ca channels in myocytes from guinea pig urinary bladder. Furthermore, the Ca 50 did not display any significant variation among different channels or cells. It was also evident that BK V,Ca channel activity could decrease in elevated [Ca 2+] i, either partially or completely. This work implies that the complete activation of BK V,Ca channels occurs with a smaller increment in [Ca 2+] s than previously expected from in vitro characterization of the Ca 2+ sensitivity of these channels. Additionally, it appears that the activity of BK V,Ca channels in situ does not strictly follow changes in near-membrane [Ca 2+].