Endogenous calcium channels in human embryonic kidney (HEK293) cells

Abstract

1 We have identified endogenous calcium channel currents in HEK293 cells. Whole cell endogenous currents (I_Sr-HEK) were studied in single HEK293 cells with 10 mM strontium as the charge carrier by the patch clamp technique. The kinetic properties and pharmacological features of I_Sr-HEK were characterized and compared with the properties of a heterologously expressed chimeric L-type calcium channel construct. 2 I_Sr-HEK activated on depolarization to voltages positive of −40 mV. It had transient current kinetics with a time to peak of 16 ± 1.4 ms (n = 7) and an inactivation times constant of 52 ± 5 ms (n = 7) at a test potential of 0 mV. The voltage for half maximal activation was −19.0 ± 1.5 mV (n = 7) and the voltage for half maximal steady-state inactivation was −39.7 ± 2.3 mV (n = 7). 3 Block of I_Sr-HEK by the dihydropyridine isradipine was not stereoselective; 1 μm (+) and (−)−isradipine inhibited the current by 30 ± 4% (n = 3) and 29 ± 2% (n = 4) respectively. (+)-Isradipine and (−)−isradipine (10 μm) inhibited I_Sr-HEK by 89 ± 4% (n = 5) and 88 ± 8% (n = 3) respectively. The 7-bromo substituted (±)-isradipine (VO2605, 10 μm) which is almost inactive on L-type calcium channels also inhibited I_Sr-HEK (83 ± 9%, n = 3) as was observed for 10 μm (−)−nimodipine (78 ± 6%, n = 5). Interestingly, 10 μm (±)-Bay K 8644 (n = 5) had no effect on the current. I_Sr-HEK was only slightly inhibited by the cone snail toxins ω-CTx GVIA (1 μm, inhibition by 17 ± 3%, n = 4) and ω-CTx MVIIC (1 μm, inhibition by 20 ± 3%, n = 4). The funnel web spider toxin ω-Aga IVA (200 nM) inhibited I_Sr-HEK by 19 ± 2%, n = 4). 4 In cells expressing I_Sr-HEK, maximum inward current densities of 0.24 ± 0.03 pA/pF and 0.39 ± 0.7 pA/pF (at a test potential of −10 mV) were estimated in two different batches of HEK293 cells. The current density increased to 0.88 ± 0.18 pA/pF or 1.11 ± 0.2 pA/pF respectively, if the cells were cultured for 4 days in serum-free medium. 5 Co-expression of a chimeric L-type calcium channel construct revealed that I_Sr-HEK and L-type calcium channel currents could be distinguished by their different voltage-dependencies and current kinetics. The current density after heterologous expression of the L-type α₁ subunit chimera was estimated to be about ten times higher in serum containing medium (2.14 ± 0.45 pA/pF) than that of I_Sr-HEK under the same conditions.