Single-channel biophysical and pharmacological characterizations of native human large-conductance calcium-activated potassium channels in freshly isolated detrusor smooth muscle cells
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Recent studies have demonstrated the importance of large-conductance Ca2+-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 Ca2+) 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 [Ca2+] from ~300 nM to Ca2+-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 [Ca2+] 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.
KeywordsLarge-conductance Ca2+-activated K+ channel Conductance Calcium Open probability Urinary bladder NS1619 Paxilline
We would like to thank MUSC Urology staff surgeons: Drs. Thomas Keane, Harry Clarke, Stephen Savage, Ross Rames, Michelle Koski, and Jonathan Picard, and Ahmed M. El-Zawahry as well as the MUSC Urology residents: Robin Bhavsar, Vinh Q. Trang, Lydia Labocetta, Elizabeth Peacock, Matthew Young, Erin Burns, Vaughan Taylor, Adonteng Kwayke, Justin Ellett, and Samuel Walker Nickles for their help with human tissue collection; and Drs. Rupal P. Soder, Kiril Hristov, Shankar Parajuli, Wenkuan Xin, Ms. Amy Smith, Mr. Serge Afeli, and Mr. Quiping Cheng for the critical evaluation of the manuscript. We are grateful to Drs. Shankar Parajuli and Kiril Hristov, and Mr. Quiping Cheng for their assistance with tissue dissection and preparation of freshly isolated DSM cells.
This study was supported by a grant from the National Institutes of Health DK084284 to Georgi V. Petkov.
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