Single-channel biophysical and pharmacological characterizations of native human large-conductance calcium-activated potassium channels in freshly isolated detrusor smooth muscle cells

Abstract

Recent studies have demonstrated the importance of large-conductance Ca(2+)-activated K(+) (BK) channels in detrusor smooth muscle (DSM) function in vitro and in vivo. However, in-depth characterization of human native DSM single BK channels has not yet been provided. Here, we conducted single-channel recordings from excised patches from native human DSM cells. Inside-out and outside-out recordings in high K(+) symmetrical solution (containing 140 mM KCl and ~300 nM free Ca(2+)) showed single-channel conductance of 215-220 pS, half-maximum constant for activation of ~+75 to +80 mV, and low probability of opening (P o) at +20 mV that increased ~10-fold at +40 mV and ~60-fold at +60 mV. Using the inside-out configuration at +30 mV, reduction of intracellular [Ca(2+)] from ~300 nM to Ca(2+)-free decreased the P o by ~85 %, whereas elevation to ~800 nM increased P o by ~50-fold. The BK channel activator NS1619 (10 μM) enhanced the P o by ~10-fold at +30 mV; subsequent application of the selective BK channel inhibitor paxilline (500 nM) blocked the activity. Changes in intracellular [Ca(2+)] or the addition of NS1619 did not significantly alter the current amplitude or single-channel conductance. This is the first report to provide biophysical and pharmacological profiles of native human DSM single BK channels highlighting their importance in regulating human DSM excitability.

Fig. 1

Voltage-dependence of native human DSM single BK channel currents recorded from excised patches in inside-out configuration. (A) Representative recordings obtained using symmetrical 140 mM KCl solution (~300 nM free Ca²⁺) illustrating increase in BK channel activity with depolarization in a single patch. C > indicates the closed channel state with inward channel openings for −20 mV and outward for +20, +40, and +60 mV. (B) Histograms for traces in (A). Plotted intervals were 0.5 s for each potential using the bins of 0.6 pA. The dotted bell shaped curves represent fitted data with the Gaussian distribution function for each state. (C) Single-channel current-voltage relationship for the single representative patch shown in (A). The mean single-channel conductance for this patch was 226 pS. (D) Single BK channel open probability (P_o) – voltage relationship for the single patch represented by the traces in (A). The V_0.5 was estimated to be +65 mV. The lower panel depicts the P_o values on the logarithmic scale illustrating very low probability of single-channel opening at −30 to +40 mV.

Fig. 2

Voltage-dependence of native human DSM single BK channel currents recorded from excised patches with outside-out configuration. (A) Representative recordings using the symmetrical 140 mM KCl solution (~300 nM free Ca²⁺) depicting increase in channel activity with depolarization in a single patch. C > indicates the closed state with inward channel openings occurring for −20 mV and their direction is outward for +20, +40, and +60 mV. (B) Histograms for traces in (A). Plotted intervals were of 0.5 sec for each potential with the bins of 0.6 pA. The dotted bell shaped curves represent fitted data with the Gaussian distribution function for each state. (C) Single-channel amplitude-voltage graph for the single patch represented in (A). The mean single-channel conductance for this patch was 231 pS. (D) Single-channel open probability (P_o)-voltage relationship for the single patch represented by traces in (A). The V_0.5 constant was estimated to be +68.9 mV. The lower panel in (D) depicts the P_o values on the logarithmic scale for the voltages in the range of −30 to +40 mV illustrating very low Po values.

Fig. 3

Comparison of biophysical properties of native human DSM single BK channel activity measured in inside-out and outside-out configurations. Shown are the summary graphs for (A) mean single-channel conductance, (B) constant for half-maximum activation (V_0.5), and (C) single-channel open probability measured at +20, +40, and +60 mV, and (D) the normalized value for open channel probability relative to the P_o at +20 mV for each patch recorded. Each data-point represents a value obtained from a separate patch recording; the horizontal line for each condition depicts the mean. The values for (A) single BK channel conductance were: 221.2 ± 3.6 pS (inside-out) and 213.8 ± 5.0 pS (outside-out); (B) V_0.5: +81.5 ± 2.6 mV (inside-out) and +76.7 ± 5.6 mV (outside-out); (C) P_o in inside-out configuration at +20, +40, and +60 mV respectively 0.010 ± 0.003, 0.065 ± 0.014, and 0.199 ± 0.030; and for P_o in outside-out configuration at +20, +40, and +60 mV respectively 0.013 ± 0.006, 0.084 ± 0.036, and 0.276 ± 0.071; (D) the normalized P_o values (+20 mV taken as 1) in inside-out configuration at +40 and +60 mV respectively 11.1 ± 1.2 and 58.8 ± 10.9; and for P_o in outside-out configuration at +40 and +60 mV respectively 9.6 ± 1.2, and 64.2 ± 12.0. The data are based on 30 inside-out single-channel patch-clamp recordings (N=13) and 15 outside-out patch-clamp recordings (N=8).

Fig. 4

Effect of removal of intracellular Ca²⁺ on native human DSM single BK channel activity. (A) A representative experiment illustrating the effect of removing intracellular Ca²⁺ (5 mM EGTA and 0 CaCl₂) using the inside-out configuration at V_m of +30 mV showing attenuation of single BK channel activity measured in a single patch. C > indicates the closed channel state and the outward currents the channel openings. (B) The plot of the P_o for the patch illustrated in (A). In this example the P_o values were 0.0089 for the control and 0.0010 for the Ca²⁺-free condition. (C) The graph of the single-channel BK current-voltage relationship for the single patch represented in (A). The mean single-channel conductance values in this example were 209.9 pS for the control and 205.3 pS for the Ca²⁺-free solution. (D) Summary of the effect of the Ca²⁺-free solution on the P_o measured at V_m of +30 mV in 12 inside-out patches (N=7). The connected filled circles depict individual experiments, and the open circles with error bars and connected line the mean values, * indicates P < 0.05 (paired Student’s t-test).

Fig. 5

Effect of elevating intracellular Ca²⁺ on native human DSM single BK channel activity. (A) A representative recording demonstrating the enhancement of single BK channel activity by increase in intracellular [Ca²⁺] from the control of ~300 nM (5 mM EGTA and 3.18 mM CaCl₂) to ~800 nM (5 mM EGTA and 4.18 mM CaCl₂) recorded in a single patch using the inside-out configuration at V_m of +30 mV. C > indicates the closed channel state. Visible are instances of multiple simultaneous channel openings especially for the solution containing ~800 nM free [Ca²⁺]. (B) Single-channel Po-voltage relationship for the single patch illustrated in (A). In this example, the P_o values were 0.0046 for the control (~300 nM Ca²⁺) and 0.4694 for ~800 nM Ca²⁺ solution. (C) Single BK channel current-voltage relationship for the patch represented in (A). The mean single-channel conductance values in this example were 254.9 pS for the control and 255.5 pS for the elevated Ca²⁺ solution. (D) Summary of the effect of the elevating free intracellular [Ca²⁺] to ~800 nM on single-channel P_o measured at +30 mV in 10 inside-out patches (N=5). The connected filled circles depict individual experiments and the open circles with error bars and connected line the mean values, # indicates P < 0.0001 (paired Student’s t-test).

Fig. 6

Pharmacological modulation of human single BK channels by the opener NS1619 and inhibitor paxilline in excised patches. (A) A representative experiment demonstrating potentiation of the single BK channel activity by the addition of NS1619 (10 µM) and blockade by the subsequent application of paxilline (500 nM in the continuous presence of 10 µM NS1619) in a single patch. The recording was obtained using the inside-out configuration at V_m of +30 mV; C > indicates the closed channel state. (B) The graph of the single-channel P_o for the single patch illustrated in (A). In this recording, the P_o values were 0.023 for the control prior to the addition and 0.165 in the presence of NS1619. (C) Single BK channel current-voltage relationship for the patch represented in (A). The mean single-channel conductance values in this example were 253.1 pS for the control and 247.1 pS in the presence of NS1619. (D) Summary of the effect of NS1619 on single BK channel P_o measured at +30 mV in 11 excised patches (N=6). The connected filled circles depict individual experiments, and the open circles with error bars and connected line the mean values, **indicates P < 0.01 (paired Student’s t-test).