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The activity of neuronal Kv7.2/Kv7.3 channels is critically dependent on PIP2 and finely modulated by cholesterol. Here, we report the crosstalk between cholesterol and PIP2 in the regulation of Kv7.2/Kv7.3 channels. Our results show that currents passing through Kv7.2/Kv7.3 channels in cholesterol-depleted cells, by acute application of methyl-β-cyclodextrin (MβCD), were less sensitive to PIP2 dephosphorylation strategies than those of control cells, suggesting that cholesterol depletion enhances the Kv7.2/Kv7.3–PIP2 interaction. In contrast, the sensitivity of Kv7.2/Kv7.3 channels to acute membrane cholesterol depletion by MβCD was not altered in mutant channels with different apparent affinities for PIP2. [ABSTRACT FROM AUTHOR]

1. In the present study, we investigated the series of events involved in the contraction of tracheal smooth muscle induced by the re-addition of Ca2+ in an in vitro experimental model in which Ca2+ stores had been depleted and their refilling had been blocked by thapsigargin. 2. Mean (±SEM) contraction was diminished by: (i) inhibitors of store-operated calcium channels (SOCC), namely 100  µmol/L SKF-96365 and 100  µmol/L 1-(2-trifluoromethylphenyl) imidazole (to 66.3 ± 4.4 and 41.3 ± 5.2% of control, respectively); (ii) inhibitors of voltage-gated Ca2+ channels CaV1.2 channels, namely 1  µmol/L nifedipine and 10  µmol/L verapamil (to 86.2 ± 3.4 and 76.9 ± 5.9% of control, respectively); and (iii) 20  µmol/L niflumic acid, a non-selective inhibitor of Ca2+-dependent Cl− channels (to 41.1 ± 9.8% of control). In contrast, contraction was increased 2.3-fold by 100 nmol/L iberiotoxin, a blocker of the large-conductance Ca2+-activated K+ (BK) channels. 3. Furthermore, contraction was significantly inhibited when Na+ in the bathing solution was replaced by N-methyl–d-glucamine (NMDG+) to 39.9 ± 7.2% of control, but not when it was replaced by Li+ (114.5 ± 24.4% of control). In addition, when Na+ had been replaced by NMDG+, contractions were further inhibited by both nifedipine and niflumic acid (to 3.0 ± 1.8 and 24.4 ± 8.1% of control, respectively). Nifedipine also reduced contractions when Na+ had been replaced by Li+ (to 10.7 ± 3.4% to control), the niflumic acid had no effect (116.0 ± 4.5% of control). 4. In conclusion, the data of the present study demonstrate the roles of SOCC, BK channels and CaV1.2 channels in the contractions induced by the re-addition of Ca2+ to the solution bathing guinea-pig tracheal rings under conditions of Ca2+-depleted sacroplasmic reticulum and inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPase. The contractions were highly dependent on extracellular Na+, suggesting a role for SOCC in mediating the Na+ influx. [ABSTRACT FROM AUTHOR]

Airway smooth muscle (ASM) contracts partly due to an increase in cytosolic Ca2+ In this work, we found that the contraction caused by histamine depends on external Na+, possibly involving nonselective cationic channels (NSCC) and the Na+/Ca2+ exchanger (NCX). We performed various protocols using isometric force measurement of guinea pig tracheal rings stimulated by histamine. We observed that force reached 53 ± 1% of control during external Na+ substitution by N-methyl-D-glucamine+, whereas substitution by Li+ led to no significant change (91 ± 1%). Preincubation with KB-R7943 decreased the maximal force developed (52.3 ± 5.6%), whereas preincubation with nifedipine did not (89.7 ± 1.8%). Also, application of the nonspecific NCX blocker KB-R7943 and nifedipine on histamine-precontracted tracheal rings reduced force to 1 ± 3%, significantly different from nifedipine alone (49 ± 6%). Moreover, nonspecific NSCC inhibitors SKF-96365 and 2-aminoethyldiphenyl borate reduced force to 1 ± 1% and 19 ± 7%, respectively. Intracellular Ca2+ measurements in isolated ASM cells showed that KB-R7943 and SKF-96365 reduced the peak and sustained response to histamine (0.20 ± 0.1 and 0.19 ± 0.09 for KB-R, 0.43 ± 0.16 and 0.47 ± 0.18 for SKF, expressed as mean of differences). Moreover, Na+-free solution only inhibited the sustained response (0.54 ± 0.25). These data support an important role for NSCC and NCX during histamine stimulation. We speculate that histamine induces Na+ influx through NSCC that promotes the Ca2+ entry mode of NCX and Cav1.2 channel activation, thereby causing contraction. [ABSTRACT FROM AUTHOR]

Airway smooth muscle contractility is due to changes in cytosolic Ca2+ and depends on external Na+. We propose that the Na+-Ca2+ exchanger (NCX) mediates Ca2+ influx secondary to intracellular Na+ increase through non-selective cationic channels (NSCC). By performing isometric force measurement of guinea-pig tracheal rings stimulated by histamine we observed that Na+ substitution by N-Methyl-D-glucamine (NMDG) produced a 47 ±7% inhibition of peak force, whereas substitution by LiCl led to no significant inhibition (9 ± 2 %). Application of NCX blocker KB-R7943 relaxed the muscle 98.96 ± 2.8 % whereas vehicle caused no relaxation (2 ± 1.3 %). Also, NSCC blockers SKF-96365 and 2-Aminoethyl diphenylborinate (2APB) caused relaxation (91 ±1% for the former and 81 ± 7% for the latter). By measuring intracellular Ca2+ changes in isolated smooth muscle cells we observed that KB-R7943 significantly decreased the fluorescence ratio in a second stimulation with histamine (0.70 ± 0.12 for control v.s. 0.49 ± 0.17 for KBR) and that SKF-96365 also decreased the fluorescence ratio (0.75 ± 0.25 for control v.s. 0.27 ± 0.10 for SKF). Altogether, these data strongly suggest an important role for the NSCC in generating a cationic current that sets the NCX in a Ca2+ entry mode and that this in turn is crucial to sustain the histamine-induced contraction. [ABSTRACT FROM AUTHOR]

The activity of neuronal Kv7.2/Kv7.3 channels is critically dependent on PIP 2 and finely modulated by cholesterol. Here, we report the crosstalk between cholesterol and PIP 2 in the regulation of Kv7.2/Kv7.3 channels. Our results show that currents passing through Kv7.2/Kv7.3 channels in cholesterol-depleted cells, by acute application of methyl-β-cyclodextrin (MβCD), were less sensitive to PIP 2 dephosphorylation strategies than those of control cells, suggesting that cholesterol depletion enhances the Kv7.2/Kv7.3-PIP 2 interaction. In contrast, the sensitivity of Kv7.2/Kv7.3 channels to acute membrane cholesterol depletion by MβCD was not altered in mutant channels with different apparent affinities for PIP 2 ., (© 2023 Walter de Gruyter GmbH, Berlin/Boston.)

Airway smooth muscle (ASM) contracts partly due to an increase in cytosolic Ca(2+). In this work, we found that the contraction caused by histamine depends on external Na(+), possibly involving nonselective cationic channels (NSCC) and the Na(+)/Ca(2+) exchanger (NCX). We performed various protocols using isometric force measurement of guinea pig tracheal rings stimulated by histamine. We observed that force reached 53 +/- 1% of control during external Na(+) substitution by N-methyl-D-glucamine(+), whereas substitution by Li(+) led to no significant change (91 +/- 1%). Preincubation with KB-R7943 decreased the maximal force developed (52.3 +/- 5.6%), whereas preincubation with nifedipine did not (89.7 +/- 1.8%). Also, application of the nonspecific NCX blocker KB-R7943 and nifedipine on histamine-precontracted tracheal rings reduced force to 1 +/- 3%, significantly different from nifedipine alone (49 +/- 6%). Moreover, nonspecific NSCC inhibitors SKF-96365 and 2-aminoethyldiphenyl borate reduced force to 1 +/- 1% and 19 +/- 7%, respectively. Intracellular Ca(2+) measurements in isolated ASM cells showed that KB-R7943 and SKF-96365 reduced the peak and sustained response to histamine (0.20 +/- 0.1 and 0.19 +/- 0.09 for KB-R, 0.43 +/- 0.16 and 0.47 +/- 0.18 for SKF, expressed as mean of differences). Moreover, Na(+)-free solution only inhibited the sustained response (0.54 +/- 0.25). These data support an important role for NSCC and NCX during histamine stimulation. We speculate that histamine induces Na(+) influx through NSCC that promotes the Ca(2+) entry mode of NCX and Ca(V)1.2 channel activation, thereby causing contraction.