Terbutaline Stereoselectively Activates Large Conductance And Ca²⁺ Dependent K⁺(BK_Ca) Channel

The ?₂-adrenergic receptor agonist terbutaline is used in the treatment of asthma.Terbutaline has one chiral centre in its structure and forms two enantiomers.Large-conductance Ca²⁺ activated K⁺ channels(BK_Ca)are densely distributed in the membrane of airway smooth muscle and have been demonstrated to play an important role in regulation of smooth muscle contractility.The present study investigated the effect of terbutaline on the single channel activity of BK_Ca channel.1.?₂ adrenergic receptors were stably overexpressed on HEK293 cells by lentiviral transduction method.Data showed that the relative m RNA expression level of ?₂ normalized to GAPDH was increased to 0.26 for HEK293-?₂ from 0.001 of normal HEK293 cells,which increased more than 200 times.2.BK_Ca channels were transiently expressed on HEK293 cells overexpressing ?₂ receptors.The single-channel recordings were performed by cell-attach patch-clamp recording mode.The dose dependent of rac-terbutaline was studied by examining the effect of rac-terbutaline with the concentration changing from 10 nM to 100 ?M on the single channel open probability of BK_Ca channel at 10 min normalized to 0 min?Po10/Po0?.The increasements of Po10/Po0 at 10 nM and 100 nM rac-ter did not reach a significant level.The value of Po10/Po0 at 1 ?M rac-ter was 4.04 ± 0.93 which was significantly larger than that of control group?1.21 ± 0.36?.Rac-ter concentrations of 10 ?M and 100 ?M induced more dramatic enlargements in Po10/Po0,which were 28.41 ± 3.71 and 30.01 ± 1.09 respectively.3.We examined the effect of R-and S-ter application with concentration of 1 ?M and 10 ?M respectively on Po of BK_Ca channel.Data showed the values of Po10/Po0 were 7.85 ± 1.31 for R-terbutaline with concentration of 1 ?M,which were significantly increased from 1.21 ± 0.36 of control group.In contrast,1 ?M S-ter did not cause any significant increase of Po10/Po0?1.06 ± 0.16?.For larger concentration,10 ?M R-terbutaline induced a great more enhancement to Po10/Po0?28.37 ± 4.98?.Different from 1 ?M,10 ?M S-ter caused a relative small but significant enlargement for Po10/Po0?2.68 ± 0.45?.4.We examined the role of ? or muscarinic receptor in the activating effects of both R-and Sterbutline by blocking ? or muscarinic receptor.Results showed that 200 nM ICI 118551 completely blocked the activating effect of 10 ?M R-terbuline by decreasing Po10/Po0 from 28.37 ± 4.98 to 1.45 ± 0.46,whereas it did not alleviate the increasing effect of 10 ?M S-ter.To investigate if the activating effect of S-ter on BK_Ca channel is mediated by muscarinic receptor,100 nM atropine was applied before patch-clamp recording.Data showed that the normalized open probability?Po10/Po0?for 10 ?M S-ter decreased from 2.68 ± 0.45 to 0.92 ± 0.28 when applied 100 nM atropine previously,which was not significantly different from that of control group?1.21 ± 0.36?.In conclusion,both R-and rac-terbutaline significantly increased the open probability of BK_Ca channel,with R-terbutaline exhibiting a prominently larger effect.The enlargement effects of R-and rac-terbutaline were blocked by ?₂ receptor antagonist ICI 118551.The Senantiomer at high concentration has a slight activity on BK_Ca channel,which may be not mediated by ?₂ adrenergic receptor but by a pathway involving muscarinic receptor.The channel activating effects of terbualine were stereoselective and the R-enantiomer was more effective.