Reply to Chadha et al.

To the editor: The letter from Drs Chadha, Greenwood, Zhong and Cole correctly points out that the drug XE991, which is commonly used as a specific inhibitor of Kv7 channels, may also inhibit other subtypes of voltage-activated K+ (Kv) channels. In an article published in 2011 in this same journal (Ng et al., 2011), Dr Greenwood and colleagues state: ‘XE991 and linopirdine block all Kv7 channels with IC50 values ∼3 µmol·L−1 (Wang et al., 2000) and at concentrations <100 µmol·L−1 are not known to have effects on any other ion channel.’ In 2010, Zhong et al. demonstrated inhibitory effects of 10 µM XE991 on cloned Kv1.2/ Kv1.5 and Kv2.1/ Kv9.3 channels in an expression system (HEK 293 cells) (Zhong et al., 2010). We had observed that 10 µM XE991 induced membrane depolarization in rat basilar artery myocytes and constriction of pressurized basilar arteries and concluded that this effect was likely due to its inhibition of Kv7 channels in the myocytes (Mani et al., 2011). What we neglected to point out in our article was that, at the resting membrane voltage of the basilar artery myocytes (∼−60 mV), the other XE991-sensitive channels (Kv1.2/ Kv1.5 and Kv2.1/ Kv9.3), would not be appreciably active because their threshold for voltage-dependent activation is more positive (∼−45 to −40 mV) (Zhong et al., 2010). Furthermore, we had previously shown that 4-aminopyridine (4-AP), a blocker of other classes of Kv channels, including Kv1.2/ Kv1.5 and Kv2.1/ Kv9.3 (Nelson and Quayle, 1995; Cox, 2005), did not significantly depolarize rat mesenteric artery myocytes (which had resting membrane voltages ∼−61 mV, similar to basilar artery myocytes) or constrict pressurized rat mesenteric arteries (Mackie et al., 2008). The specificity of XE991 as a blocker of Kv7 channels is supported by our finding that knocking down expression of Kv7.5 channels in A7r5 vascular smooth muscle cells completely eliminated the XE991-sensitive currents (Mani et al., 2009). We would assert that at resting membrane voltages of −60 to −45 mV, Kv7 channels are the only Kv channels that have an appreciable open probability under physiological conditions and therefore the effects of XE991 (figure 3 of Mani et al., 2011) that we observed can reasonably be attributed to inhibition of Kv7 channels. Vascular myocytes express a wide variety of ion channels, making it a challenge to isolate the contribution of a particular subset of channels. In some cases, the biophysical properties of the channels can be used to effectively isolate them from other channels using patch clamp electrophysiology. We have utilized the perforated patch configuration, 5 s voltage steps from −4 mV holding potential, and an external solution supplemented with gadolinium chloride to effectively isolate Kv7 currents in vascular myocytes over the physiological voltage range between −65 and −20 mV. Gadolinium chloride blocks Ca2+ influx that might activate Ca2+-activated K+ channels (KCa) and also shifts the voltage dependence of activation of 4-AP-sensitive Kv channels ∼15 mV in the positive direction (Mani et al., 2011). The perforated patch configuration is essential because we find that the Kv7 currents run down significantly within a few minutes in a ruptured patch configuration (L.I. Brueggemann and K.L. Byron, unpublished experiments.). The native vascular Kv7 currents measured with our recording conditions have electrophysiological characteristics of cloned Kv7 channels, including kinetics of deactivation, voltage-dependence of activation, etc. (Brueggemann et al., 2011). We have also shown that these currents are fully inhibited by pharmacological Kv7 channel blockers (XE991 or linopirdine), but insensitive to pharmacological blockers of other classes of vascular K+ channels, including drugs that inhibit KCa, KATP and other subtypes of Kv channels (Mackie et al., 2008). The inhibitory effect of XE991 on currents recorded at voltages ≤−20 mV was irreversible in every vascular smooth muscle preparation we have tested (L.I. Brueggemann and K.L. Byron, unpubl. obs.), whereas the enhancement of the currents by drugs such as flupirtine and celecoxib was fully reversed on washout of drugs (Brueggemann et al., 2007; 2009;). Wladyka and Kunze similarly found that inhibition of the Kv7 -mediated M-currents in nodose neurons was sustained on washout of XE991, while the inhibition of other subtypes of Kv currents was rapidly reversed (Wladyka and Kunze, 2006). Thus, the irreversibility of block by XE991 further supports our contention that the currents we record are mediated by Kv7 channels. By contrast, the strategies employed by Zhong et al. to record Kv7 currents in vascular myocytes have yielded a mix of several currents, only a small fraction of which is blocked (reversibly) by XE991 or linopirdine (Zhong et al., 2010). This may be attributed to use of a ruptured patch configuration, short (≤500 ms) voltage steps from a hyperpolarized holding potential, and test voltages at which other types of ion channels are predominant. The contribution of Kv7 channels is inferred by subtracting the majority of the signal to reveal the XE991- or linopirdine-sensitive current, typically at +20 to +40 mV. At these voltages, other types of K+ channels, including 4-AP-sensitive Kv channels, are likely to overwhelm the smaller Kv7 currents (Mackie et al., 2008). The results of Zhong et al. indicate that both XE991 and linopirdine can inhibit these 4-AP-sensitive channels at positive voltages. In summary, we respectfully suggest that the electrophysiological and pharmacological approaches we have employed in our studies enable us to conclude with reasonable confidence that Kv7 channels have an important functional role in determining basilar artery myocytes' resting membrane voltage. We remain convinced that the effects of XE991 on membrane voltage and basilar artery constriction that we reported in our article can be attributed primarily to its actions as an inhibitor of Kv7 channels.