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Endogenous calcium channels in human embryonic kidney (HEK293) cells
Author(s) -
Berjukow Stanislav,
Döring Frank,
Froschmayr Monika,
Grabner Manfred,
Glossmann Hartmut,
Hering Steffen
Publication year - 1996
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1996.tb15463.x
Subject(s) - hek 293 cells , isradipine , dihydropyridine , chemistry , biophysics , voltage dependent calcium channel , calcium , calcium channel , patch clamp , pharmacology , biochemistry , receptor , medicine , biology , organic chemistry
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 μ m (+) and (−)−isradipine inhibited the current by 30 ± 4% ( n = 3) and 29 ± 2% ( n = 4) respectively. (+)‐Isradipine and (−)−isradipine (10 μ m ) inhibited I Sr‐HEK by 89 ± 4% ( n = 5) and 88 ± 8% ( n = 3) respectively. The 7‐bromo substituted (±)‐isradipine (VO2605, 10 μ m ) which is almost inactive on L‐type calcium channels also inhibited I Sr‐HEK (83 ± 9%, n = 3) as was observed for 10 μ m (−)−nimodipine (78 ± 6%, n = 5). Interestingly, 10 μ m (±)‐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 μ m , inhibition by 17 ± 3%, n = 4) and ω‐CTx MVIIC (1 μ m , 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 α 1 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.