Pharmacological characterization of small-conductance Ca(2+)-activated K(+) channels stably expressed in HEK 293 cells

Abstract

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. The bee venom peptide apamin blocked hSK1 as well as rSK2 with IC(50) values of 3.3 nM and 83 pM, respectively. 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. The potent small molecule blockers showed little differentiation 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. The nicotinic acetylcholine receptor antagonist d-tubocurarine was found to block hSK1 and rSK2 with IC(50) values of 27 microM and 17 microM when measured at +80 mV. The inhibition by d-tubocurarine was voltage-dependent with increasing affinities at more hyperpolarized potentials. 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 microM when measured at +80 mV. 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.

Figure 1

Current-voltage relations and Ca²⁺-dependent activation of whole-cell SK currents. The whole-cell currents recorded after application of voltage-ramps (−100 to +100 mV, 200 ms duration) to the SK expressing HEK 293 cell lines are shown as a function of the ramp potential for a hSK1 expressing cell (a) and a rSK2 expressing cell (b). The two traces in each panel were obtained with 140 mM Na⁺ plus 4 mM K⁺ or with 144 mM K⁺ in the bath solutions. (c) is the current at −80 mV and at +80 mV measured from voltage ramps applied to a hSK1 expressing cell and plotted as a function of time. The arrow indicates the time at which an experiment would normally start.

Figure 2

Inhibition of hSK1 by apamin. The inhibition of SK currents were studied in dose-response experiments as the one shown in (a) and in single-dosely experiments as shown in (c). The whole-cell currents measured at +80 mV are plotted as a function of time. In (a) the bath solution was shifted to solutions with the indicated concentrations of apamin at the times marked by arrows and in (c) the presence of 3 nM apamin in the bath solution is indicated by the bar. The dotted line in (a) indicates the estimated baseline during the time of apamin application. (b) and (d) illustrate the calculation of IC₅₀ values for the two experiments shown in (a) and (c). In (b) the symbols are the percentage of current remaining plotted as a function of the apamin concentration. The solid curve was obtained after applying a Hill fitting procedure to the data-points. In (d) the symbols are the current measured during application of apamin whereas the curve is the fitted curve obtained from equation (2) in Methods.

Figure 3

Inhibition of rSK2 by apamin. (a) Dose-response experiment for apamin on a rSK2 expressing HEK 293 cell depicted as the whole-cell current measured at +80 mV as a function of time. The bath solution was changed at the times indicated by the arrows and the values listed are the apamin concentration in the extracellular solution in nM. (b) Current versus time plot for a single-dose experiment in which 300 pM apamin was applied to a rSK2 expressing cell during the time indicated by the bar.

Figure 4

Voltage-dependent block of SK channels by d-tubocurarine. The whole-cell currents as a function of the ramp potential for a hSK1 expressing cell (a) and a rSK2 expressing cell (b). Traces obtained before (ctrl) and after addition of d-tubocurarine at the concentrations indicated (in μM) are shown. The 400 samples recorded during the ramps after block by 30 μM d-tubocurarine were divided by the samples recorded from the corresponding control ramps and these values are depicted as a function of the ramp potential in (c) and (d) to illustrate the voltage-dependency of the block by d-tubocurarine.

Figure 5

Block of hSK1 by bicuculline methiodide. The fast block of hSK1 current by bicuculline methiodide is shown as a function of time. The bath solutions were changed at the times indicated by arrows and the values are the bicuculline methiodide concentrations in the extracellular solution in μM. The dotted line is the estimated baseline during the time of drug application.