Pharmacological activation and inhibition of Slack (Slo2.2) channels
Introduction
The significance of potassium (K+) conductances in the regulation of neuronal excitability and their involvement in neuronal pathologies has been well documented (Hille, 2001, Levitan and Kaczmarek, 2002). K+ channels encoded by the Slack (Sequence like a calcium-activated K channel) (Slo2.2) gene are gated by intracellular Na+, and Slack channels are located in the brain regions (Joiner et al., 1998, Bhattacharjee et al., 2002, Yuan et al., 2003) reported to possess sodium-activated K+ (KNa) channels (Egan et al., 1992, Dryer, 1994, Bhattacharjee and Kaczmarek, 2005). Nevertheless, the physiological significance of these KNa conductances in the central nervous system (CNS) is not yet fully understood. The discovery of potent blockers and activators of Slack channels may therefore lead to a better understanding of the roles these channels play under physiological and/or patho-physiological circumstances.
Other than the closely related KNa channel subunit Slick (Bhattacharjee et al., 2003), the channel with the highest similarity to Slack is the Ca2+-activated potassium channel Slo, with ∼7% identity (Joiner et al., 1998). Slack has several domains in its long carboxy terminal region that show clear homology to Slo. Nevertheless, the pharmacology of the Slack current is quite distinct from that of Slo channels. Slack channels are insensitive to the large conductance calcium-activated K+ (BKCa or maxi-K) channel blocking agent iberiotoxin and the BKCa channel opener NS-1619, but are inhibited by millimolar (10 mM) concentrations of tetraethylammonium ions (TEA) (Joiner et al., 1998, Bhattacharjee et al., 2003).
In this study we have characterized the actions of quinidine and bepridil, two compounds that have been shown to inhibit KNa channels in ventricular myocytes (Mori et al., 1998, Li et al., 1999), on the properties of Slack channels expressed in oocytes and mammalian cells. Quinidine and bepridil are mixed ion channel blockers and have been used as antiarrhythmic drugs. They are known to block several types of K+ channels, as well as other types of voltage-dependent ion channels, in cardiac myocytes, neurons and other cell types (Kehl, 1991, Zilberter et al., 1994, Lesage et al., 1996, Sato et al., 1996, Chouabe et al., 1998, Leonoudakis et al., 1998, Reyes et al., 1998, Kobayashi et al., 2001, Yumoto et al., 2004). In contrast to these blocking agents, there have been no previous descriptions of activators or openers of either native KNa channels or of the Slack channel. We now report that bithionol, a commercially available bis-phenol anti-parasitic compound (Enzie and Colglazier, 1960, Barr et al., 1965), is an effective opener of Slack channels. The pharmacological characterization of Slack currents in transfected cells will allow a more rigorous comparison of these currents to KNa currents in native neurons, and may also represent an important first step in the development of more potent and specific KNa modulators.
Section snippets
Stable cell line expressing the Slack channel
The SlackHA.pCDNA3 construct, containing the full-length wild type Slack sequence, was used to transfect HEK-293 cells. Transfection was performed using the SuperFect Transfection Reagent (QIAGEN Inc., Valencia, CA). The stable Slack-expressing HEK cell line was confirmed by patch-clamp recordings and Western blot. These HEK cells were cultured in a modified low sodium DMEM medium supplemented with 10% fetal bovine serum and penicillin-streptomycin (Invitrogen Inc, Carlsbad, CA).
Electrophysiological recordings from Slack-expressing Xenopus oocytes
Two-electrode
Bepridil inhibits macroscopic Slack currents
We first examined the actions of bepridil on Slack currents expressed in Xenopus oocytes. As reported previously (Joiner et al., 1998), oocytes injected with Slack mRNA responded to depolarizing voltage pulses with large (2–5 μA) outwardly rectifying currents. In all cases, uninjected oocytes displayed relatively low-amplitude currents only at very high depolarizing potentials (reflective of endogenous Ca2+-activated chloride current), and were not significantly affected by any of the compounds
Discussion
The significance of voltage-gated potassium conductances in the regulation of neuronal excitability and their involvement in neuronal pathologies has been well documented (Shieh et al., 2000, Levitan and Kaczmarek, 2002). Slack is a ligand-gated K+ channel that is activated by intracellular Na+ ions and is abundantly expressed in those mammalian CNS regions reported to possess KNa channels (Joiner et al., 1998, Bhattacharjee et al., 2002, Bhattacharjee and Kaczmarek, 2005).
Since the first
Acknowledgements
This work was supported by the NIH Grants DC-01919 and NS42202 to Dr. L.K. Kaczmarek. Dr. B. Yang is supported by a Heritage Affiliate Postdoctoral Fellowship from the American Heart Association.
References (39)
- et al.
Potentiation of the antimicrobial activity of bithionol
J. Pharm. Sci.
(1965) - et al.
For potassium channels, sodium is the new potassium
Trends Neurosci.
(2005) Na+-activated K+ channels: a new family of large-conductance ion channels
Trends Neurosci.
(1994)- et al.
Na+-activated K+ channels are widely distributed in rat CNS and in Xenopus oocytes
Brain Res.
(1992) - et al.
Regulation of the timing of MNTB neurons by short-term and long-term modulation of potassium channels
Hearing Res.
(2005) - et al.
Inhibitory effect of bepridil on hKv1.5 channel current: comparison with amiodarone and E-4031
Eur. J. Pharmacol.
(2001) - et al.
Cloning and expression of a novel pH-sensitive two pore domain K+ channel from human kidney
J. Biol. Chem.
(1998) - et al.
Block of Na+ channel by bepridil in isolated guinea-pig ventricular myocytes
Eur. J. Pharmacol.
(1996) Electrophysiology of calcium antagonists
J. Mol. Cell. Cardiol.
(1987)- et al.
The sodium-activated potassium channel is encoded by a member of the Slo gene family
Neuron
(2003)
Localization of the Slack potassium channel in the rat central nervous system
J. Comp. Neurol.
Slick (Slo2.1), a rapidly-gating sodium-activated potassium channel inhibited by ATP
J. Neurosci.
A fuzzy subsarcolemmal space for intracellular Na+ in cardiac cells?
Cardiovasc. Res.
HERG and KvLQT1/IsK, the cardiac K+ channels involved in long QT syndromes, are targets for calcium channel blockers
Mol. Pharmacol.
The effects of external cations and ouabain on the intracellular sodium activity of sheep heart Purkinje fibres
J. Physiol.
Preliminary trials with bithionol against tapeworm infections in cats, dogs, sheep, and chickens
Am. J. Vet. Res.
Effects of channel modulators on cloned large-conductance calcium-activated potassium channels
Mol. Pharmacol.
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches
Pflugers Arch.
Ionic Channels of Excitable Membranes
Cited by (69)
Potassium channelopathies associated with epilepsy-related syndromes and directions for therapeutic intervention
2023, Biochemical PharmacologyK<inf>Na</inf>1.1 gain-of-function preferentially dampens excitability of murine parvalbumin-positive interneurons
2022, Neurobiology of DiseaseCitation Excerpt :However, it is now thought that subcellular localization of the channel near microdomains of elevated sodium, maintained by voltage-gated sodium channels and the sodium‑potassium pump, allows KNa1.1 to impact neuronal excitability as a delayed rectifier conductance, contributing to the resting membrane potential, afterhyperpolarization current, and action potential threshold (Gray and Johnston, 2021; Wallen et al., 2007). In particular, the frequency of action potentials during high-frequency stimulation is fine-tuned by the level of KNa1.1 activity (Yang et al., 2006). Since the first reported association with epilepsy pathogenesis in 2012 (Barcia et al., 2012), pathogenic variants in KCNT1 are increasingly reported with a broad phenotypic spectrum from early-onset developmental and epileptic encephalopathies including epilepsy of infancy with migrating focal seizures (EIMFS) to later-onset focal epilepsy and sleep-related hypermotor epilepsy (SHE) (Borlot et al., 2020; Heron et al., 2012; Ishii et al., 2013; Kingwell, 2012; Lim et al., 2016; McTague et al., 2013; McTague et al., 2018; Ohba et al., 2015).
Targeting K<inf>Na</inf>1.1 channels in KCNT1-associated epilepsy
2021, Trends in Pharmacological SciencesCitation Excerpt :KCNT1-related epilepsies are intractable, and conventional therapies only temporarily alleviate symptoms. Antiarrhythmic drugs quinidine and bepridil are efficacious at inhibiting WT and pathogenic variant KNa1.1 channels expressed in Xenopus oocytes and mammalian cells [14,54,55,60,61]. A third antiarrhythmic drug, clofilium, has also been found to inhibit WT channels in vitro [62].