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. 2009 Sep;158(2):521-31.
doi: 10.1111/j.1476-5381.2009.00332.x. Epub 2009 Jul 23.

Inhibition of vascular calcium-gated chloride currents by blockers of KCa1.1, but not by modulators of KCa2.1 or KCa2.3 channels

Affiliations

Inhibition of vascular calcium-gated chloride currents by blockers of KCa1.1, but not by modulators of KCa2.1 or KCa2.3 channels

W R Sones et al. Br J Pharmacol. 2009 Sep.

Abstract

Background and purpose: Recent pharmacological studies have proposed there is a high degree of similarity between calcium-activated Cl(-) channels (CaCCs) and large conductance, calcium-gated K(+) channels (K(Ca)1.1). The goal of the present study was to ascertain whether blockers of K(Ca)1.1 inhibited calcium-activated Cl(-) currents (I(ClCa)) and if the pharmacological overlap between K(Ca)1.1 and CaCCs extends to intermediate and small conductance, calcium-activated K(+) channels.

Experimental approaches: Whole-cell Cl(-) and K(+) currents were recorded from murine portal vein myocytes using the whole-cell variant of the patch clamp technique. CaCC currents were evoked by pipette solutions containing 500 nM free [Ca(2+)].

Key results: The selective K(Ca)1.1 blocker paxilline (1 microM) inhibited I(ClCa) by approximately 90%, whereas penitrem A (1 microM) and iberiotoxin (100 and 300 nM) reduced the amplitude of I(ClCa) by approximately 20%, as well as slowing channel deactivation. Paxilline also abolished the stimulatory effect of niflumic acid on the CaCC. In contrast, an antibody against the Ca(2+)-binding domain of murine K(Ca)1.1 had no effect on I(ClCa) while inhibiting spontaneous K(Ca)1.1 currents. Structurally different modulators of small and intermediate conductance calcium-activated K(+) channels (K(Ca)2.1 and K(Ca)2.3), namely 1-EBIO, (100 microM); NS309, (1 microM); TRAM-34, (10 microM); UCL 1684, (1 microM) had no effect on I(ClCa).

Conclusions and implications: These data show that the selective K(Ca)1.1 blockers also reduce I(ClCa) considerably. However, the pharmacological overlap that exists between CaCCs and K(Ca)1.1 does not extend to the calcium-binding domain or to other calcium-gated K(+) channels.

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Figures

Figure 2
Figure 2
Effects of penitrem A and iberiotoxin on IClCa. (A) The effect of 10 µM penitrem A (Pen) on IClCa. (A)i shows representative currents recorded in the absence and presence of penitrem A (5 min, 1 µM). (A)ii and (A)iii show the effect of penitrem on I–V relationships and reversal potential of IClCa. (A)iv shows the effect of penitrem A at different voltages with the corresponding currents and amplitudes. (B) The same variables as in (A), but for 300 nM iberiotxin (IbTx). Each point or bar is the mean data from at least four cells (bars showing SEM). *P < 0.05, **P < 0.01 and ***P < 0.001 for paired Student's t-test comparisons between data acquired before and after application of the channel modulator.
Figure 1
Figure 1
Effects of paxilline on IClCa. (A) A sample trace showing IClCa recorded in the absence and after 5 min application of 10 µM paxilline (Px). IClCa was evoked by step depolarization to +70 mV followed by repolarization to −80 mV. (B) The I–V relationship of the voltage and time-dependent current evoked by stepping to voltages ranging between −80 and +120 mV in the absence and presence of paxilline. (C) The reversal potential of the evoked current was not affected by paxilline; voltage protocol is shown below the graph. (D) The concentration–response relationship for paxilline inhibition of IClCa recorded at +70 mV. Each point is the mean data from at least four cells (bars showing SEM). *P < 0.05 and **P < 0.01 for paired Student's t-test comparisons between data acquired before and after application of paxilline.
Figure 3
Figure 3
Effects of tamoxifen and 17β-oestradiol on IClCa. Modulation by tamoxifen (Tx, 10 µM; A) and 17β-oestradiol (Oest, 10 µM; B) of IClCa. (A)i and (B)i show sample traces, in the same cell, control IClCa and IClCa in the presence of the channel modulator, recorded at +70 mV followed by repolarizing to −80 mV. (A)ii and (B)ii show effects on the I–V relationships of the voltage and time-dependent current evoked by stepping to voltages ranging between −80 and +120 mV. In (A)iii and (B)iii, the current evoked immediately on repolarization to voltages ranging from −100 to +40 mV, is shown. The bar charts [(A)iv and (B)iv] detail different aspects of IClCa evoked by a depolarizing step to +70 mV followed by a repolarizing step to −80 mV; control currents and those evoked in the presence of modulator are shown. Each point or bar represents the mean data from at least four cells (bars showing SEM). *P < 0.05, **P < 0.01 and ***P < 0.001 for paired Student's t-test comparisons between data acquired before and after application of the channel modulator.
Figure 4
Figure 4
Co-application of niflumic acid and paxilline. (A) A sample trace showing in the same cell, inhibition of control IClCa by the initial application of niflumic acid (100 µM) followed by co-application of paxilline (1 µM). (B) Another sample trace demonstrating the reverse application; paxilline followed by co-application of niflumic acid. IClCa was evoked by step depolarization to +70 mV followed by repolarization to −80 mV.
Figure 6
Figure 6
Lack of effect of anti-KCa 1.1 antibody on IClCa. (A) A comparison of IClCa recorded in a cell 10 min after achieving whole-cell access in a control cell (left-hand trace) or one dialysed with the anti-KCa 1.1 antibody (1:200: right-hand trace). (B) The lack of effect evoked by the anti-KCa 1.1 antibody (1:200) within the patch pipette when compared to control over a period of 10 min. Points represent late current evoked on stepping to +70 mV at a time period after acquiring whole-cell access, expressed as normalized current evoked immediately upon breaking the seal (relative to the initial current recorded at time = 0). (C) and (D) The lack of effect of the antibody on the I–V relationship of the voltage and time-dependent current evoked by stepping to voltages ranging between −80 and +120 mV, and the current evoked immediately on repolarization from +80 mV to voltages ranging from −100 to +40 mV respectively. Each point represents mean data from at least three cells (bars showing SEM).
Figure 5
Figure 5
Modulation of spontaneous transient outward currents (STOCs) by anti-KCa 1.1 antibody. (A–C) Representative recordings of STOCs at 0 mV under control conditions, after 5 min application of 10 nM iberiotoxin (B) or after 10 min intracellular dialysis with an anti-KCa 1.1 antibody (1:200, C). Bar chart (D) demonstrates the absolute changes in mean peak STOC amplitude over a 1 min period before and 5 min after application of iberiotoxin or following dialysis with anti-KCa 1.1 antibody in the absence of iberiotoxin. (E) The absolute changes in total area encapsulated within STOC and baseline over a similar time period. Each bar is the mean data from at least three cells (bars showing SEM). **P < 0.01 for paired Student's t-test; comparisons between data acquired before and after application of the channel modulator.
Figure 7
Figure 7
Effects of KCa 2.1 and KCa 2.3 modulators on IClCa. Modulation by 1-EBIO (100 µM; A), NS309 (1 µM; B), TRAM-34 (10 µM; C) and UCL 1684 (1 µM; D) is shown in this figure with a sample trace (left) showing in the same cell, control IClCa and IClCa evoked in the presence of a KCa 2.1 or KCa 2.3 modulator by depolarization to +70 mV followed by repolarizing to −80 mV. Middle and right graphs portray the I–V relationships of the voltage and time-dependent current evoked by stepping to voltages ranging between −80 and +120 mV, and the current evoked immediately on repolarization to voltages ranging from −100 to +40 mV, respectively; control currents and currents evoked in the presence of relevant modulator are shown. Each point represents the mean data from at least three cells (bars showing SEM).

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