4-Chloro-benzo[F]isoquinoline (CBIQ) activates CFTR chloride channels and KCNN4 potassium channels in Calu-3 human airway epithelial cells

Abstract

1 Calu-3 cells have been used to investigate the actions of 4-chloro-benzo[F]isoquinoline (CBIQ) on short-circuit current (SCC) in monolayers, whole-cell recording from single cells and by patch clamping. 2 CBIQ caused a sustained, reversible and repeatable increase in SCC in Calu-3 monolayers with an EC50 of 4.0 microm. Simultaneous measurements of SCC and isotopic fluxes of 36Cl- showed that CBIQ caused electrogenic chloride secretion. 3 Apical membrane permeabilisation to allow recording of basolateral membrane conductance in the presence of a K+ gradient suggested that CBIQ activated the intermediate-conductance calcium-sensitive K(+)-channel (KCNN4). Permeabilisation of the basolateral membranes of epithelial monolayers in the presence of a Cl- gradient suggested that CBIQ activated the Cl(-)-channel CFTR in the apical membrane. 4 Whole-cell recording in the absence of ATP/GTP of Calu-3 cells showed that CBIQ generated an inwardly rectifying current sensitive to clotrimazole. In the presence of the nucleotides, a more complex I/V relation was found that was partially sensitive to glibenclamide. The data are consistent with the presence of both KCNN4 and CFTR in Calu-3. 5 Isolated inside-out patches from Calu-3 cells revealed clotrimazole-sensitive channels with a conductance of 12 pS at positive potentials after activation with CBIQ and demonstrating inwardly rectifying properties, consistent with the known properties of KCNN4. Cell-attached patches showed single channel events with a conductance of 7 pS and a linear I/V relation that were further activated by CBIQ by an increase in open state probability, consistent with known properties of CFTR. It is concluded that CBIQ activates CFTR and KCNN4 ion channels in Calu-3 cells.

Figure 1

Shows the responses (mean values±s.e.) of Calu-3 monolayers to CBIQ, 7,8-benzoquinoline and 5,6-benzoquinoline in the absence (closed circles) and in the presence (open circles) of IBMX (100 μM, bs). Each individual curve, indicated by n values, was analysed to give an EC₅₀value and the mean values±s.e. for these are shown adjacent to each curve. The structures of the three compounds are shown. An inset shows a maintained response to CBIQ, 20 μM given at the arrow.

Figure 2

Shows the effect of acetazolamide (100 μM, bs) and bumetanide (20 μM, bl) on the responses to CBIQ (50 μM, bs) in two Calu-3 monolayers. Note that the extent of the inhibition to either inhibitor is dependent on the order in which they are given.

Figure 3

(a) and (b) illustrate a paired experiment with Calu-3 monolayers. Both monolayers were initially treated on the apical surface with nystatin, 200 μg ml⁻¹, in the presence of an apical to basolateral potassium gradient and in the absence of permeant anions. In (a) after nystatin, CBIQ (10 μM, bs) was added, causing a current increase that was reversed by the addition of ChTX (50 nM, bl), with a smaller extra inhibition by 293B (20 μM, bl). In the control experiment (b), the inhibitors were added after nystatin. When CBIQ (10 μM, bs) was added subsequently, a small response was produced compared to that seen in (a). In (c) and (d), the cumulative data for six paired experiments are given. The response to CBIQ, 10 μM, shown in (d) was significantly smaller than the value given in (c). (P<0.001, n=6).

Figure 4

(a) and (b) illustrate a paired experiment with Calu-3 monolayers, one of which was pre-exposed to ChTX, 50 nM and 293B, 20 μM (both added basolaterally). Each monolayer was then treated with CBIQ, 20 μM (bs). (c) and (d) show, respectively, the maximal SCC increases and the charge transfers during 10 min for three paired experiments without (open columns) and with blocking agents (closed columns). The increases in SCC and charge transfer were significantly greater in the absence of the blocking agents (P<0.001 and <0.002, respectively).

Figure 5

Shown are representative examples of responses of Calu-3 monolayers subjected to an apical to basolateral chloride gradient. In (a), the basolateral membrane was depolarised with PGK solution, while in (b), the basolateral surface was permeabilised by treatment with nystatin, as described in the Methods section. CBIQ, 10 μM (bs) and DPC (bl) were added as indicated.

Figure 6

Whole-cell recording in Calu-3 cells. (a) Shows a representative whole-cell recording in the absence of ATP and GTP and demonstrating the effect of CBIQ, 10 μM, followed by clotrimazole, 30 μM. (b) Shows the I/V relationship corresponding to the traces depicted in (a). Note the hyperpolarizing shift in the reversal potential. Cumulative data from six experiments are given in Table 2. (c) Is a representative whole-cell recording showing the effects of CBIQ, 10 μM, with subsequent addition of glibenclamide, 100 μM, in the presence of ATP and GTP. (d) Shows the I/V relationship corresponding to the traces depicted in (c). Cumulative data from several experiments are given in Table 2.

Figure 7

Representative recording from an excised inside-out patch (260 s) of a Calu-3 cell. (a). No channel activity is recorded under control conditions at −60 mV until CBIQ, 10 μM, is added, indicated by the arrow. Subsequent addition of clotrimazole, 30 μM, as indicated, completely eliminated the channel activity. Arrows, to the left of the traces, designate the closed channel state. (b). The all points histogram was derived from 60 s of the recording shown in (a), starting at a point 20 s after the addition of CBIQ. The unitary current amplitude (i) was calculated as the difference between the mean peak currents of a two peak Gaussian fit.

Figure 8

Single channel properties of the CBIQ activated K⁺-channels in excised inside-out patches of Calu-3 cells. In the presence of CBIQ, 10 μM, the patch was clamped from –80 to 80 mV in 20 mV steps, each lasting 10 s. Representative recordings at each step are shown. Arrows to the left of the traces designate the closed state. (b) Shows the I/V relationship with inward rectification (n=3).

Figure 9

Recording from a cell-attached patch of a Calu-3 cell. Shown is a representative recording (110 s) demonstrating a relatively low channel activity under control conditions when clamped at a negative pipette potential (−V_PIP) of 60 mV. The addition of CBIQ, 10 μM (at the arrow), significantly stimulated channel activity. NP_O values were 0.52±0.51 (n=3) in control conditions, increasing to 2.63±0.38 (n=3) (P<0.03) after CBIQ. Arrows to the left of the traces indicate the closed state.

Figure 10

Single channel properties of the CBIQ activated channels in attached patches of Calu-3 cells. (a). In the presence of CBIQ, the membrane was clamped at negative pipette potentials (−V_PIP) from −80 to 80 mV in 20 mV increments, each lasting 5 s. Representative recordings from each step are shown. Arrows to the left of the traces indicate the closed state. (b) The I/V relationship of the channel collected from three experiments as in (a) is shown in (b). Mean values and s.e. are shown. From a linear fit of all points, the channel is shown to have a unitary conductance of 6.7±0.5 pS.