Elsevier

Neuropharmacology

Volume 51, Issue 4, September 2006, Pages 896-906
Neuropharmacology

Pharmacological activation and inhibition of Slack (Slo2.2) channels

https://doi.org/10.1016/j.neuropharm.2006.06.003Get rights and content

Abstract

The Slack (Sequence like a calcium-activated K channel) (Slo2.2) gene is abundantly expressed in the mammalian brain and encodes a sodium-activated K+ (KNa) channel. Although the specific roles of Slack channel subunits in neurons remain to be identified, they may play a role in the adaptation of firing rate and in protection against ischemic injury. In the present study, we have generated a stable cell line expressing the Slack channel, and have analyzed the pharmacological properties of these channels in these cells and in Xenopus oocytes. Two known blockers of KNa channels, bepridil and quinidine, inhibited Slack currents in a concentration-dependent manner and decreased channel activity in excised membrane patches. The inhibition by bepridil was potent, with an IC50 of 1.0 μM for inhibition of Slack currents in HEK cells. In contrast, bithionol was found to be a robust activator of Slack currents. When applied to the extracellular face of excised patches, bithionol rapidly induced a reversible increase in channel opening, suggesting that it acts on Slack channels relatively directly. These data establish an important early characterization of agents that modulate Slack channels, a process essential for the experimental manipulation of Slack currents in neurons.

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.

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