Pharmacological characterization of small‐conductance Ca²⁺‐activated K⁺ channels stably expressed in HEK 293 cells

Abstract

1 Three genes encode the small-conductance Ca 2+ -activated K + channels (SK channels). We have stably expressed hSK1 and rSK2 in HEK 293 cells and addressed the pharmacology of these subtypes using whole-cell patch clamp recordings. 2 The bee venom peptide apamin blocked hSK1 as well as rSK2 with IC 50 values of 3.3 nM and 83 pM, respectively. 3 The pharmacological separation between the subtypes was even more prominent when applying the scorpion peptide blocker scyllatoxin, which blocked hSK1 with an IC 50 value of 80 nM and rSK2 at 287 pM. 4 The potent small molecule blockers showed little di erentiation between the channel subtypes. The bis-quinolinium cyclophane UCL 1684 blocked hSK1 with an IC 50 value of 762 pM and rSK2 at 364 pM. The antiseptic compound dequalinium chloride blocked hSK1 and rSK2 with IC 50 values of 444 nM and 162 nM, respectively. 5 The nicotinic acetylcholine receptor antagonist d-tubocurarine was found to block hSK1 and rSK2 with IC 50 values of 27 mM and 17 mM when measured at +80 mV. The inhibition by dtubocurarine was voltage-dependent with increasing a nities at more hyperpolarized potentials. 6 The GABA A receptor antagonist bicuculline methiodide also blocked hSK1 and rSK2 in a voltage-dependent manner with IC 50 values of 15 and 25 mM when measured at +80 mV. 7 In conclusion, the pharmacological separation between SK channel subtypes expressed in mammalian cells is too small to support the notion that the apamin-insensitive afterhyperpolarization of neurones is mediated by hSK1.

Ca2+-activated K+ Channels in Human Leukemic Jurkat T Cells

“…5C). A similar inward rectification was also observed for rSK2 expression in HEK 293 cells (37). A few minutes after break-in to the whole cell configuration, the hSK2 current has the tendency to hSK2 with rSK2, aSK2, hSK3, rSK3, hSK1, and rSK1.…”

“…Recent studies revealed that Ca 2ϩ gating involves the constitutive association of calmodulin with a C-terminal domain of SK channel ␣ subunits (31)(32)(33)(34). Some cloned isoforms of SK channels are blocked by apamin and by d-tubocurarine (dTC), a plant alkaloid (29,(35)(36)(37). The residues Asp 341 and Asn 368 on either side of the deep pore of the ␣ subunit were shown to be the primary determinants of apamin and dTC sensitivity (35 In this study, we cloned from human Jurkat T cells a 2.5-kb full-length cDNA, hSK2, encoding the human isoform of SK2 channels.…”

Regional Differences in Distribution and Functional Expression of Small-Conductance Ca2+-Activated K+Channels in Rat Brain

“…This, along with the lower apamin sensitivity of homomeric SK channels, may seem to support the hypothesis that SK1 underlies the apamin-insensitive sAHP. However, this idea seems still difficult to reconcile with the observation that the hippocampal sAHP seems to be entirely resistant to high concentrations of apamin, which completely block the homomultimeric SK1 channels tested in different expression systems (Shah and Haylett, 2000a;Strobaek et al, 2000;Grunnet et al, 2001b). This hypothesis might, however, be rescued if there exist auxiliary (␤) subunits that substantially alter the toxin sensitivity of the SK channel complex, such as the BK-␤4 subunits have been found to do for BK channels (Meera et al, 2000).…”

“…It was therefore suggested that the sAHP is mediated by SK1 channels. However, the findings that the sAHP is resistant to apamin (up to 1 M; Lancaster and Nicoll, 1987;Storm, 1989;Stocker et al, 1999), whereas SK1 homomeric channels are blocked by this toxin (Shah and Haylett, 2000a;Strobaek et al, 2000;Grunnet et al, 2001a), have, together with other pharmacological and kinetic discrepancies, cast doubt on the proposed causal relationship between SK1 and the sAHP (Shah and Haylett, 2000b;Shah et al, 2001;Faber and Sah, 2002). Meanwhile, a complex alternative splicing pattern of mouse and rat SK1 genes have been discovered (Shmukler et al, 2001), with some splice variants lacking the calmodulin-binding domain.…”

Anxiolytic Effects of Maxipost (BMS-204352) and Retigabine via Activation of Neuronal Kv7 Channels

“…As previously described (Strøbaek et al, 2000), K i values were calculated based on current versus time plots and the kinetics of drug-induced reduction in the current. This is fitted with the nonequilibrium version of the Michaelis-Menten equation:…”