Endogenous calcium channels in human embryonic kidney (HEK293) cells

Abstract

We have identified endogenous calcium channel currents in HEK293 cells. Whole cell endogenous currents (ISr-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 ISr-HEK were characterized and compared with the properties of a heterologously expressed chimeric L-type calcium channel construct.2 Isr-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 ISrHEK by the dihydropyridine isradipine was not stereoselective; 1 gM (+) and (-isradipine inhibited the current by 30+4% (n=3) and 29+2% (n=4) respectively. (+)-Isradipine and (-)-isradipine (10 giM) inhibited ISr-HEK by 89 + 4% (n =5) and 88 + 8% (n = 3) respectively. The 7-bromo substituted (±)-isradipine (V02605, 10 giM) which is almost inactive on L-type calcium channels also inhibited ISr-HEK (83+9%, n=3) as was observed for 10 kIM (-)-nimodipine (78+6%, n=5). Interestingly, 10 gIM (±)-Bay K 8644 (n =5) had no effect on the current. ISr-HEK was only slightly inhibited by the cone snail toxins wo-CTx GVIA (1 gM, inhibition by 17 + 3%, n = 4) and Cw-CTx MVIIC(1 gM, inhibition by 20 + 3%, n = 4). The funnel web spider toxin w-Aga IVA (200 nM) inhibited ISr-HEK by 19+2%, n=4). 4 In cells expressing ISr-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 ISr-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 oa, subunit chimera was estimated to be about ten times higher in serum containing medium (2.14 + 0.45 pA/pF) than that of ISr HEK under the same conditions.

The Na+/Ca2+Exchange Inhibitor 2-(2-(4-(4-Nitrobenzyloxy)phenyl)ethyl)isothiourea Methanesulfonate (KB-R7943) Also Blocks Ryanodine Receptors Type 1 (RyR1) and Type 2 (RyR2) Channels

“…In addition, the KB-R7943 inhibition on caffeine-stimulated Ca 2ϩ release in HEK 293 cells stably transfected with RyR1 supports the hypothesis of a direct effect on RyR. Because HEK 293 cells do not express Ca v 1.2 protein (Berjukow et al, 1996), an indirect effect of KB-R7943 on RyR mediated by CaV1.2 does not seem to be essential. However, we cannot rule out an additional effect of this inhibitor on dihydropyridine receptor voltage sensor activity.…”

Nonspecific Cation Current Associated with Native Polycystin-2 in HEK-293 Cells

“…Despite many observations of a TEAsensitive delayed rectifier K ϩ current, not all authors agree about the presence of native chloride conductances (26,27). A voltage-dependent Ca 2ϩ current, sensitive to dihydropyridine but different from L-type, has been described (28). Occasionally, findings of a voltage-dependent TTX-sensitive Na ϩ current have also been reported (29), although HEK-293 cells endogenously express the auxiliary ␤1A-subunit of voltage-gated sodium channel but not the pore-forming ␣ one (30).…”

Voltage-dependent Changes of TRPV6-mediated Ca2+ Currents

“…The I-V relationship showed a negative reversal potential, most likely caused by a basal Cl Ϫ conductance present in these cells (15). The inward current at potentials around 30 mV may arise through the activation of voltage-dependent channels, which are expressed in this cell line (16) and are not deactivated at such negative potentials (Fig. 1B, top).…”