Biophys J, October 1998, p. 1774-1782, Vol. 75, No. 4

In Situ Characterization of the Ca²⁺ Sensitivity of Large Conductance Ca²⁺-Activated K⁺ Channels: Implications for Their Use as Near-Membrane Ca²⁺ Indicators in Smooth Muscle Cells

Departamento de Bioquímica, CINVESTAV-IPN, México D. F. 07000, México

The Ca²⁺ sensitivity of large conductance Ca²⁺- 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 [Ca²⁺] ([Ca²⁺]_i). The latter were induced by ionomycin, an electroneutral Ca²⁺ ionophore. At 0 mV, the Hill coefficient (n_H) and the [Ca²⁺]_i to attain half of the maximal BK_V,Ca channel activity (Ca₅₀) 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 [Ca²⁺] ([Ca²⁺]_s) and the bulk [Ca²⁺]_i, indicated by fura-2. High Hill numbers in the activation by Ca²⁺ 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 Ca²⁺ cooperativity is the normal situation for BK_V,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 BK_V,Ca channel activity could decrease in elevated [Ca²⁺]_i, either partially or completely. This work implies that the complete activation of BK_V,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 BK_V,Ca channels in situ does not strictly follow changes in near-membrane [Ca²⁺].

Biophys J, October 1998, p. 1774-1782, Vol. 75, No. 4
© 1998 by the Biophysical Society   0006-3495/98/10/1774/09  $2.00

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