Characteristics of a human N-type calcium channel expressed in HEK293 cells.
The human alpha 1B-1 alpha 2b beta 1-2 Ca2+ channel was stably expressed in HEK293 cells producing a human brain N-type voltage-dependent calcium channel (VDCC). Whole cell voltage-clamp electrophysiology and fura-2 based microfluorimetry have been used to study its characteristics. Calcium currents (ICa) recorded in transfected HEK293 cells were activated at potentials more depolarized than -20 mV with peak currents occurring at approx + 10 mV in 5 mM extracellular CaCl2. ICa and associated rises in intracellular free calcium concentrations ([Ca2+]i) were sensitive to changes in both the [Ca2+]o and holding potential. Steady-state inactivation was half maximal at a holding potential of -60 mV. Ba2+ was a more effective charge carrier than Ca2+ through the alpha 1B-1 alpha 2b beta 1-2 Ca2+ channel and combinations of both Ba2+ and Ca2+ as charge carriers resulted in the anomalous mole fraction effect. Ca2+ influx into transfected HEK293 cells was irreversibly inhibited by omega-conotoxin-GVIA (omega-CgTx-GVIA; 10 nM-1 microM) and omega-conotoxin-MVIIA; 100 nM-1 microM) whereas 1 microM) whereas no reductions were seen with agents which block P or L-type Ca2+ channels. The inorganic ions, gadolinium (Gd3+), cadmium (Cd2+) and nickel (Ni2+) reduced the ICa under voltage-clamp conditions in a concentration-dependent manner. The order of potency of the three ions was Gd3+ > Cd2+ > Ni2+. These experiments suggest that the cloned and expressed alpha 1B-1 alpha 2b beta 1-2 Ca2+ channel subunits form channels in HEK293 cells that exhibit properties consistent with the activity of the native-N-type VDCC previously described in neurons.
Lilly Research Centre Ltd, Windlesham, Surrey, U.K.
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Bleakman
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Bowman
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Bath
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Brust
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Johnson
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Deal
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Miller
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Ellis
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Harpold
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Hans
D002104:Cadmium / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000235:genetics*; D002478:Cells, Cultured / Q000187:drug effects; D004594:Electrophysiology; D016257:Fura-2; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects*; D009532:Nickel / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000494:pharmacology; D013997:Time Factors; D020866:omega-Conotoxin GVIA
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D010455:Peptides; D002104:Cadmium; D009532:Nickel; D020866:omega-Conotoxin GVIA; D002118:Calcium; D016257:Fura-2