In the human airway, activation of basolateral KCa3.1 promotes transepithelial Cl– secretion across WT and corrected F508del HBEs (21, 22, 24, 25, 35). Further, apical BKCa plays a vital role in maintaining the ASL volume (26–28). Thus, we determined whether CFTR correctors would alter K+ channel function when acutely applied to HBEs.

Initially, we determined the effect of the current standard of care (SOC) C2 CFTR corrector, VX-445, on BKCa function in primary WT CFTR HBEs grown at an air-liquid interface on Transwell filters. Studies were carried out in Ussing chambers using a 125:5 mM K+ gradient (basal to apical) to measure K+ secretion across the epithelia, as previously described (20). Note that our solutions result in a large apical-to-basolateral Na+ gradient. As shown in Figure 1A, amiloride was used to inhibit the basal Na+ absorption, resulting in an inwardly directed current, consistent with K+ secretion across the apical membrane. Subsequent addition of VX-445 (10 μM) stimulated a large, slowly developing increase in inward current that was completely inhibited by the specific BKCa blocker paxilline (10 μM). This result demonstrates that VX-445 stimulates K+ secretion across the apical membrane of HBEs. Consistent with activation of BKCa, VX-445 decreased transepithelial resistance (Rte) from 521 ± 57 Ω∙cm2 in the presence of amiloride to 283 ± 18 Ω∙cm2, while addition of paxilline increased Rte to 1,155 ± 109 Ω∙cm2 (n = 30). As shown in Figure 1B, a distinct C2 corrector, VX-659 (10 μM), similarly stimulated a paxilline-sensitive K+ current. More recently, Vertex Pharmaceuticals developed a next-generation C2 CFTR corrector, VX-121, which is currently undergoing clinical trials (36). As shown in Figure 1C, subsequent to amiloride, VX-121 (10 μM) also stimulated a large, paxilline-sensitive K+ secretory current. As above, VX-121 decreased Rte from 453 ± 49 Ω∙cm2 in the presence of amiloride to 248 ± 17 Ω∙cm2, while addition of paxilline increased Rte to 691 ± 68 Ω∙cm2 (n = 29), consistent with BKCa activation and inhibition, respectively. We next determined whether other components of ETI would stimulate K+ secretion across HBEs. Neither the C1 corrector VX-661 (Figure 1D) nor the CFTR potentiator VX-770 (Figure 1E) induced K+ secretion, whereas subsequent addition of VX-445 or VX-121 stimulated K+ secretion, respectively. To further confirm that this effect was due to apical BKCa activation, we determined whether the currents induced by VX-445 and VX-121 could be inhibited by an additional selective BKCa inhibitor, IBTX. As a 37–amino acid peptide, IBTX cannot cross the apical membrane. As shown, both the VX-445–induced (Figure 1F) and VX-121–induced (Figure 1G) K+ currents were inhibited by IBTX (300 nM), verifying activation of BKCa. Similar results were observed in 6 experiments each for VX-445 and VX-121. Given these surprising results, we determined the effect of a known BKCa opener, NS1619, on K+ secretion across HBEs. As shown in Figure 1H, 10 μM NS1619 failed to stimulate K+ secretion, though the subsequent addition of 50 μM NS1619 stimulated a substantial K+ secretory response. Further addition of VX-445 (10 μM) stimulated an additional K+ secretory response that was completely inhibited by paxilline. The average responses are shown in Figure 1I, having a rank order of VX-121 > VX-445 > VX-659 (*P < 0.01).

VX-445, VX-659, and VX-121 stimulate BKCa currents across WT CFTR HBEs. Currents were recorded with a 125:5 mM K+ gradient (basolateral to apical). (A–C) Subsequent to amiloride, short-circuit current (Isc) was increased by the C2 CFTR correctors VX-445 (A, 10 μM), VX-659 (B, 10 μM), and VX-121 (C, 10 μM). (D and E) In contrast, the C1 CFTR corrector VX-661 (D, 10 μM) and the CFTR potentiator VX-770 (E, 10 μM) failed to increase Isc. Subsequent addition of either VX-445 (D) or VX-121 (E) stimulated Isc. In all experiments, the current was completely blocked by the specific BKCa inhibitor paxilline (10 μM). (F and G) Additional studies verified that both the VX-445–induced (F) and the VX-121–induced (G) currents were inhibited by the additional specific BKCa blocker IBTX (300 nM). (H) Subsequent to amiloride, 10 μM NS1619 failed to stimulate Isc, while further addition of 50 μM NS1619 induced a marked increase. This response was further increased by VX-445 (10 μM) and inhibited by paxilline. (I) Average responses (mean ± SEM, *P < 0.01; 1-way ANOVA) are represented as the change in K+ current (ΔIK). The magnitude of the K+ current was calculated as described in Methods. Nine donors were used in these studies. Experimental replicates are indicated in parentheses above each data set.

While the most parsimonious explanation for our results is that C2 correctors potentiate apical BKCa, we cannot rule out a potential role for the basolateral membrane in this response. Therefore, the effects of VX-445 and VX-121 on apical membrane K+ currents were assessed after permeabilization of the basolateral membrane with nystatin (200 μM), as previously described (37). Formation of the nystatin pore is visualized in Figure 2, A and B, as a positive shift in baseline current to a new stable plateau. Subsequent addition of either VX-445 (Figure 2A) or VX-121 (Figure 2B) stimulated paxilline-sensitive K+ currents that were indistinguishable from those in the absence of permeabilization. Consistent with our intact monolayers, the response to VX-121 was significantly greater than the response to VX-445 (Figure 2C; *P < 0.01). These results verify that C2 CFTR correctors potentiate apical BKCa in WT CFTR–expressing HBEs.

VX-445 and VX-121 stimulate BKCa currents across WT CFTR HBEs following permeabilization of the basolateral membrane with nystatin. Currents were recorded with a 125:5 mM K+ gradient (basolateral to apical). Subsequent to amiloride, nystatin (200 μM) was added to the basolateral membrane. Following establishment of a new stable current, both VX-445 (A, 10 μM) and VX-121 (B, 10 μM) stimulated an increase in IK. (C) Average ΔIK (mean ± SEM) for VX-445 and VX-121 (*P < 0.01; unpaired t test). ΔIK was calculated as the change in IK between the current in the presence of nystatin and the peak response to VX-445 or VX-121. Three donors were used in these studies. Experimental replicates are indicated in parentheses above each data set.

We next determined the effects of varying the concentration of VX-445 and VX-121 on K+ secretion to assess concentration dependence. As shown in Figure 3A, VX-445 stimulated K+ secretion at low-micromolar concentrations, and exhibited a steep concentration dependence between 1 and 10 μM (Figure 3C). Likewise, VX-121 stimulated K+ secretion at low-micromolar concentrations. However, VX-121 appeared more potent, producing visible responses at 0.3 and 1 μM (Figure 3B). As above, paxilline inhibited the K+ currents stimulated by VX-445 and VX-121 (Figure 3, A and B). Summary data for these studies are provided in Figure 3C. As the response to VX-121 approached saturation at 10 μM, we were able to fit these data to the Hill equation (Figure 3D), and obtained an apparent EC50 of 4.4 μM with a Hill coefficient of 3. In contrast, VX-445 did not saturate at 10 μM, and thus we were not able to obtain a reliable estimate of the EC50 for this molecule. We did not routinely go to the next higher half-log concentration (30 μM), as this is above the concentration of VX-445 achieved in plasma (Cmax = 15 μM; ref. 38). Further, in 3 experiments in which we applied 30 μM VX-445, the subsequent addition of paxilline resulted in an increase in Rte of only 154 Ω∙cm2, whereas in 8 experiments carried out on the same day in which 10 μM VX-445 was added, the subsequent addition of paxilline increased Rte by 654 Ω∙cm2. As one possibility to explain this result is an overall decrease in Rte, we did not pursue these higher concentrations further. Importantly, our results demonstrate that VX-445 stimulates K+ secretion in the range of concentrations known for VX-445 in both plasma and cells of patients with CF (see Discussion).

Concentration dependence of VX-445 and VX-121 stimulation of K+ current across WT CFTR HBEs. (A) Subsequent to amiloride, VX-445 induced a concentration-dependent increase in K+ current that was inhibited by paxilline. (B) Subsequent to amiloride, VX-121 induced a concentration-dependent increase in K+ current that was inhibited by paxilline. (C) Average increase in K+ current for each concentration of VX-121 and VX-445 (mean ± SEM). Note that for VX-445 there are different numbers of experiments for each concentration, as not all concentrations were used in each experiment. (D) Data from C for VX-121 were fit to the Hill equation, giving an EC50 of 4.4 μM with a Hill coefficient of 3.0 (R2 = 0.8). Three donors were used in these studies. Experimental replicates are indicated in parentheses above each data set.

Next, we determined whether CFTR correctors stimulate K+ secretion across homozygous F508del CFTR HBEs. As shown in Figure 4A, following amiloride, the current SOC C1 corrector, VX-661 (10 μM), had no effect on K+ secretory current. However, subsequent addition of the current SOC C2 corrector, VX-445 (10 μM), stimulated a sustained K+ secretory current, akin to the response of WT CFTR HBEs. Further, initial addition of VX-445 (10 μM) induced a K+ secretory current that was not further increased by VX-661 (Figure 4B). Consistent with activation of an ionic conductance, VX-445 decreased Rte from 221 ± 7 Ω∙cm2 in the presence of amiloride to 146 ± 6 Ω∙cm2 (P < 0.001, n = 28), and this was increased to 491 ± 24 Ω∙cm2 (P < 0.001, n = 28) following paxilline addition. As in WT CFTR HBEs, VX-659 (10 μM) stimulated K+ secretion across F508del CFTR HBEs (Figure 4C). Similarly, the next-generation C2 CFTR corrector VX-121 (10 μM) stimulated a large, paxilline-sensitive K+ secretory current (Figure 4D). Again, this was accompanied by a decrease in Rte from 203 ± 9 Ω∙cm2 in the presence of amiloride to 140 ±4 Ω∙cm2 (P < 0.001, n = 28). Inhibition of BKCa by paxilline increased Rte to 441 ± 29 Ω∙cm2 (P < 0.001, n = 28), as expected. Finally, we determined whether VX-809 (lumacaftor), a first-generation C1 corrector, would affect transepithelial K+ currents across F508del CFTR HBEs. As shown in Figure 4E, VX-809 (10 μM) had no effect on K+ current, while the subsequent addition of VX-121 (10 μM) again stimulated a paxilline-sensitive K+ current. We used the peak response to thapsigargin (1 μM) as the gold standard for activation of BKCa (Figure 4F), as we have previously shown that thapsigargin stimulates K+ secretion across HBEs under the conditions used here (20). The average response of K+ secretion to each CFTR corrector across F508del CFTR HBEs is shown in Figure 4G. Based on these results, we conclude that C1 correctors fail to activate BKCa, whereas the C2 CFTR correctors activate BKCa with a relative potency of VX-121 > VX-445 >> VX-659.

Effect of CFTR correctors on BKCa currents in F508del CFTR HBEs. Currents were recorded with a 125:5 mM K+ gradient (basolateral to apical) from uncorrected F508del HBEs. (A) Subsequent to amiloride, VX-661 (10 μM) failed to stimulate K+ secretion, while the further addition of VX-445 (10 μM) stimulated a paxilline-sensitive K+ secretory current. (B) After amiloride, VX-445 (10 μM) induced K+ secretion, whereas the addition of VX-661 (10 μM) failed to stimulate K+. (C) VX-659 (10 μM) induced a paxilline-sensitive K+ current. (D) VX-121 (10 μM) stimulated a paxilline-sensitive K+ current. (E) VX-809 (10 μM) failed to stimulate K+ secretion, while further addition of VX-121 (10 μM) induced an increase in K+ current. (F) Thapsigargin (1 μM) stimulated a transient K+ current. (G) Average ΔIK values (mean ± SEM) for each compound evaluated (*P < 0.05; #P < 0.01; ND, not different; 1-way ANOVA). Six donors were used in these studies. Experimental replicates are indicated in parentheses above each data set.

As CFTR is activated by cAMP/PKA, which also modulates BKCa, it seems likely that CFTR and BKCa are simultaneously activated during cAMP-mediated agonist addition (39, 40). Thus, we determined whether forskolin stimulates K+ secretion across F508del CFTR HBEs and whether this affects the ability of VX-445 to stimulate K+ secretion. Initially, we verified that forskolin stimulates K+ secretion across F508del CFTR HBEs. As shown in Figure 5A, subsequent to amiloride, forskolin (10 μM) stimulated a rapid inward current followed by a sustained paxilline-sensitive plateau. As HBEs express Kv7.1, Kv7.3, and Kv7.5 (KCNQ) channels (35, 41, 42), which are also activated by cAMP/PKA, we determined whether these channels were contributing to the K+ secretory response elicited by forskolin. The pan-Kv7.X inhibitor XE-991 (10 μM) produced only a modest decrease in K+ secretory current induced by forskolin, suggesting that Kv7.X channels are not responsible for the K+ secretory current observed. To verify that the initial transient increase in K+ current was also due to BKCa activation, we used paxilline to inhibit the baseline K+ current induced by amiloride (Figure 5B), thereby validating that the inward current revealed by amiloride block of Na+ absorption was indeed due to K+ secretion. As shown in Figure 5B, preaddition of paxilline completely eliminated the forskolin response, verifying that both the peak and plateau currents were due to BKCa activation. Finally, we determined whether the VX-445 or forskolin responses were affected by the prior addition of the other compound. As shown in Figure 5C, VX-445 (10 μM) stimulated a further increase in K+ current subsequent to forskolin. However, in comparison with preaddition of VX-445 (Figure 5D), this response was decreased in magnitude (Figure 5E). Similarly, while prior addition of VX-445 (10 μM) did not affect the peak response to forskolin, the extent of the plateau phase was reduced (Figure 5, D and E).

Effect of forskolin and VX-445 on BKCa currents in F508del CFTR HBEs. (A) Subsequent to amiloride, forskolin (10 μM) stimulated a paxilline-sensitive increase in K+ secretion, which is recognized as an initial transient spike followed by a sustained increase in Isc. Addition of XE-991 (10 μM) had little effect on the remaining current. (B) Subsequent to amiloride and paxilline, forskolin (10 μM) failed to stimulate K+ secretion. (C) Following forskolin, VX-445 (10 μM) induced a further increase in K+ secretion, which was paxilline sensitive. (D) VX-445 (10 μM) stimulated a sustained increase in K+ secretory current, which was further increased by forskolin. Subsequent addition of paxilline completely inhibited this K+ secretory current. (E) Average responses (mean ± SEM) to forskolin and VX-445 either alone or after addition of the previous agonist (#P < 0.01; unpaired ANOVA). Three donors were used in these studies. Experimental replicates are indicated in parentheses above each data set.

We next considered whether chronic exposure to VX-445 would affect BKCa currents. First, however, we determined whether the effect of VX-445 was readily reversible. This is important, as we could not maintain the filters in VX-445 during the experiment since this would simply recapitulate our demonstrated acute effects, even in the absence of a chronic effect. On the other hand, if VX-445 is readily reversible, any chronic effects may be lost when the filters are bathed in our apical/basolateral solutions. As shown in Figure 6, following stimulation of K+ current with VX-445 (10 μM), we carried out 6 complete solution exchanges of the apical and basal chambers (during a break in recording). Following this wash, BKCa short-circuit currents rapidly returned to pre-potentiated levels. Subsequent addition of paxilline (10 μM) inhibited the remaining current, as above. In 3 separate experiments, washout of VX-445 resulted in a reduction in current averaging 90% ± 6%. Thus, any effects of chronic exposure would be difficult to interpret, using these methods, and were not further pursued.

Effect of VX-445 on K+ current can be completely washed out in Ussing chambers. Following stimulation of K+ current by VX-445 (10 μM), both membranes were washed via 6 complete solution exchanges (noted by a break in recording). Following washout, the K+ current rapidly returned toward the pre–VX-445 current level. Subsequent addition of paxilline (10 μM) completely inhibited the remaining current. In 3 experiments, the average reduction in K+ current following washout of VX-445 was 90% ± 6%.

The simplest explanation for our HBE results is that the C2 CFTR correctors VX-659, VX-445, and VX-121 directly activate apical membrane BKCa. To assess this, we determined whether VX-445 and VX-121 potentiate BKCa during whole-cell patch-clamp recordings from HEK cells (HEK-BK) heterologously expressing the pore-forming α subunit of BKCa (αBKCa). For these studies, the cell was clamped at –80 mV and pulsed in 20 mV increments to +80 mV. As shown for a single cell (Figure 7, A–C), following establishment of a stable baseline (Figure 7A), VX-445 (5 μM) induced a significant increase in outward current (Figure 7B) that was completely inhibited by paxilline (Figure 7C). Similar studies were carried out using VX-121. As shown for a single cell (Figure 7, D–F), VX-121 (5 μM) potentiated αBKCa currents, which were blocked by paxilline. The average current-voltage (I-V) relationships for control and 1 μM, 5 μM, and 10 μM VX-445, as well as paxilline, are shown in Figure 7G. Both 5 and 10 μM VX-445 significantly increased BKCa current density across the range of voltages tested. The average I-V relationships for 1 μM, 5 μM, and 10 μM VX-121 are shown in Figure 7H. Similarly to VX-445, our results show that 5 and 10 μM VX-121 significantly potentiated αBKCa current density.

Effect of CFTR correctors on whole-cell BKCa currents heterologously expressed in HEK cells. (A) Control whole-cell recording. Voltage was stepped between –80 to +80 mV in 20 mV increments. (B) Whole-cell recording from the same cell following stimulation with 5 μM VX-445. (C) Paxilline (2 μM) completely blocked BKCa current. (D) Control whole-cell current recording. (E) Whole-cell current recording from the same cell following stimulation with 5 μM VX-121. (F) Paxilline (2 μM) completely blocked BKCa current. (G) Average whole-cell current-voltage (I-V) relationships (mean ± SEM) for control (n = 21) and 1 μM (n = 8), 5 μM (n = 14), and 10 μM (n = 11) VX-445 (*P < 0.05, **P < 0.01, ***P < 0.005; paired t test). Paxilline was added in the presence of VX-445 to illustrate lack of outward K+ currents in these cells when BKCa is blocked (n = 4). (H) Average whole-cell I-V relationships (mean ± SEM) for control (n = 14) and 1 μM (n = 6), 5 μM (n = 8), and 10 μM (n = 6) VX-121 (*P < 0.05, **P < 0.01, ***P < 0.005; paired t test). Complete inhibition by paxilline confirms a lack of additional VX-121–potentiated currents in this stable cell line.

To demonstrate that the potentiation of αBKCa by VX-445 during whole-cell recording is a direct effect on the channel, we performed excised, inside-out patch-clamp recordings on HEK-BK cells. As shown in Figure 8A, excised patches often contained many αBKCa channels. In this case, mean currents were determined in the absence and presence of VX-445. As shown in Figure 8A, 1 μM VX-445 produced a small increase in total current, and this was further increased by 10 μM VX-445. Channel activity was dramatically reduced following perfusion of 0 Ca2+. As shown in Figure 8B, both 1 and 10 μM VX-445 produced a significant increase in mean current (P < 0.05). In additional inside-out patches, small numbers of channels were observed (<5), such that individual opening and closing events could be realized, thereby allowing us to determine both single-channel current amplitude (i) and open probability (Po). As shown for one experiment in Figure 8C, both 1 and 10 μM VX-445 increased channel Po, as evidenced by the increased frequency of discrete channel opening events (Figure 8, C and D). The average change in Po for 4 patches is shown in Figure 8H, with 10 μM VX-445 inducing a 10-fold increase in Po. All-point histograms for the recording shown in Figure 8C during control (Figure 8E) and 1 μM (Figure 8F) and 10 μM (Figure 8G) VX-445 demonstrated no significant change in i (Figure 8I). In total, our patch-clamp studies demonstrate that the current SOC C2 corrector, VX-445, directly potentiates αBKCa via an increase in Po, likely explaining the K+ secretion observed across HBEs.

Effect of VX-445 in excised patches. Excised patch-clamp recordings were carried out in symmetric K+ and voltage-clamped to +40 mV. (A) Both 1 μM and 10 μM VX-445 increased current in a patch expressing many channels. The current was reversed by addition of 0 Ca2+. (B) Average mean current (pA, mean ± SEM) for excised patches containing numerous BKCa channels for control (n = 5) and 1 μM (n = 4) and 10 μM (n = 5) VX-445. (C) Both 1 μM and 10 μM VX-445 increased current in a patch expressing αBKCa such that individual channel openings could be observed. (D) Expanded view of trace shown in C, where individual BKCa channel open and closed events can be observed in magnified current traces corresponding to the parent trace (dashed lines and i, ii, and iii). (E–G) All-point histograms from the recording shown in C such that single-channel current amplitude can be determined, as indicated by the distance between the dotted lines placed at the peak of each curve, which represent a given open state. (H) Average Po (mean ± SEM) calculated for 4 experiments, as shown in C. (I) Average single-channel amplitude for 4 experiments (*P < 0.05; paired ANOVA).

Having verified potentiation of αBKCa in HEK-BK cells, we determined whether C2 correctors potentiate BKCa currents in primary undifferentiated, nonpolarized HBEs via whole-cell patch clamp. As shown in Figure 9A, HBEs exhibited an outwardly rectified current that was potentiated by VX-445 (10 μM; Figure 9B) and subsequently completely inhibited by paxilline (Figure 9C), verifying expression of BKCa and potentiation by VX-445. To further validate the identity of these VX-445–potentiated currents, we used another canonical blocker of the BKCa channel, IBTX. As shown in Figure 9F, IBTX inhibited the VX-445–potentiated K+ currents (Figure 9, D and E). The average I-V relationships for 9 experiments are shown in Figure 9G. Interestingly, some primary HBEs, surveyed by whole-cell patch clamp, had low-level BKCa expression, in which single-channel activity was observed (Figure 9, H and I). In Figure 9H, under control conditions (left array of traces), little or no channel activity was observed at +20 or +40 mV, while at +60 mV clear channel activity was seen. This is consistent with the voltage dependence of BKCa channels. However, in the presence of VX-445 (10 μM; right array of traces) multiple channel openings were observed at all voltages. Paxilline completely abrogated channel activity in this cell (Figure 9H, right bottom trace). In a separate cell (Figure 9I), no channel activity was observed at +40, +60, or +80 mV under control conditions (left array of traces). Subsequent addition of VX-445 (10 μM) induced individual channel events, which were silenced with 300 nM IBTX. These data validate BKCa potentiation in HBEs, which consist of several cell types that can potentially express BKCa (43, 44), including CFTR-expressing ionocytes (45).

VX-445 potentiates paxilline- and IBTX-sensitive currents in WT CFTR HBEs. (A) Control whole-cell recording from primary HBEs during voltage steps from –80 to +80 mV in 20 mV increments. (B and C) Effect of VX-445 (B, 10 μM) and paxilline (C, 1 μM) on the cell shown in A. (D) Control whole-cell recording from primary HBEs. (E and F) This current was potentiated by VX-445 (E, 10 μM) and inhibited by IBTX (F, 300 nM). (G) Mean I-V (mean ± SEM) for control (squares), 10 μM VX-445, and 1 μM paxilline from 9 experiments. (H) Whole-cell recording from primary HBEs where individual single-channel openings can be observed at +20, +40, and +60 mV. Control traces (left) exhibit fewer channel openings when compared with those observed in the presence of 10 μM VX-445 (right) or when 1 μM paxilline was added to inhibit BKCa activity (bottom right). (I) Whole-cell recording from primary HBEs where individual single-channel openings cannot be observed at +40, +60, and +80 mV in control recordings (left). Addition of VX-445 (10 μM, right) induced channel activity, which was inhibited by paxilline (1 μM, bottom right).

Our data demonstrate that C2 CFTR correctors directly potentiate BKCa channels, resulting in K+ secretion across WT and F508del CFTR–expressing HBEs. As BKCa is widely expressed throughout the body (46, 47), it is important to determine whether the current SOC C2 corrector, VX-445, modulates the function of additional tissues where BKCa is expressed. Initially, we determined the effect of VX-445 on vascular reactivity, as activation of BKCa hyperpolarizes vascular smooth muscle, resulting in vasorelaxation (48, 49). To assess the effect of VX-445 on vasoreactivity, mouse mesenteric arteries were preconstricted with the prostaglandin mimetic U46619 (1 × 10−7 to 5 × 10−7 M), and the ability of VX-445 to induce vasorelaxation was assessed. As shown in Figure 10A for a single mesenteric artery, following U46619-induced vasoconstriction, VX-445 induced vasorelaxation in a concentration-dependent manner. The average response is shown in Figure 10B (blue), with near-complete vasorelaxation achieved at 10 μM VX-445. This effect was partially attenuated by paxilline (Figure 10B, red), demonstrating VX-445 alters vasoreactivity in a BKCa-dependent manner.

Effect of VX-445 on vasoreactivity in mouse mesenteric artery. (A) Recording of force in millinewtons (mN) over time from a single mesenteric artery showing preconstriction with the prostaglandin mimetic U46619 (1 × 10−7 to 5 × 10−7 M), after which the ability of increasing concentrations of VX-445 to induce vasorelaxation was assessed. We added 0 Ca2+ at the end to determine maximal vasorelaxation. (B) Average responses to VX-445 under control conditions (blue line, n = 10) and following preincubation with paxilline (10 μM) for 15 minutes (red line, n = 6). The effect of VX-445 was partially reversed by paxilline, verifying a role for BKCa. *Statistical difference between VX-445 and VX-445 + paxilline by 2-way ANOVA with P < 0.003 by post hoc Holm-Šidák multiple-comparison test. Data are shown as mean ± SEM.

In the nervous system, BKCa channels are a key component of the fast afterhyperpolarization, which is an important contributor to neuronal firing frequency (50, 51). Indeed, channelopathies involving both gain and loss of function of BKCa have been reported (52, 53). Thus, we determined whether VX-445 and VX-121 alter neuronal excitability. To accomplish this, current-clamp patch-clamp recordings were performed on primary E18 rat hippocampal and cortical neurons, and action potential firing frequency was monitored. As shown for 2 separate recordings from spontaneously firing hippocampal neurons (Figure 11, A and B), VX-445 induced a concentration-dependent decrease in action potential firing frequency, which was reversible upon washout. Indeed, as shown in Figure 11B, action potential firing frequency could be repeatedly inhibited by 10 μM VX-445. The average changes in firing frequency (in hertz) for 2.5 μM (n = 5), 5 μM (n = 9), and 10 μM (n = 5) VX-445 are shown in Figure 11C. As shown in Figure 11D, VX-121 (5 μM) similarly reduced action potential firing frequency in a primary hippocampal neuron, with the average change for 5 experiments shown in Figure 11E. Finally, we determined whether VX-445 would similarly alter the action potential firing frequency in primary cortical neurons to begin to assess the generalizability of our results. In contrast to hippocampal neurons, current injection was required to induce action potential firing in cortical neurons, under our recording conditions. As shown in Figure 11F, after current injection (delineated by the step change in voltage at the initiation of the trace), action potentials were observed. Subsequent addition of VX-445 (2.5 and 5 μM) induced a significant reduction in action potential firing frequency that was poorly washed out. The average change in firing frequency for 11 separate neurons is shown in Figure 11G. These results clearly demonstrate that VX-445 and VX-121 alter neuronal excitability, the implications of which are discussed below.

Effect of CFTR correctors on action potential firing in primary rat hippocampal and cortical neurons. (A) Effect of 2.5, 5, and 10 μM VX-445 on action potential firing in a spontaneously firing hippocampal neuron. (B) Hippocampal neuron demonstrating the reversible inhibition of action potential firing by 10 μM VX-445. (C) Average action potential firing frequency, in hertz, for experiments carried out as in A and B for control (n = 9) and 2.5 μM (n = 5), 5 μM (n = 9), and 10 μM (n = 5) VX-445 (*P < 0.05, **P < 0.01; paired t test). (D) Effect of VX-121 (5 μM) on a spontaneously firing hippocampal neuron. (E) Average action potential firing frequency for control and VX-121 (n = 5) (**P < 0.01). (F) Effect of VX-445 on action potential firing frequency in a primary cortical neuron. Action potentials were induced by current injection (step change in voltage). (G) Average action potential firing frequency (hertz) for control and 2.5 μM and 5 μM VX-445 in cortical neurons (n = 11 for all conditions, *P < 0.05; paired ANOVA).