The human α1B-1β1-2Ca2+ 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 occuring 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 α1B-1α2bβ1-2 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 ω-conotoxin-GVIA (ω-CgTx-GVIA; 10 nM-1 μM) and cu-conotoxin-MVIIA (ω-CmTx-MVHA; 100 nM-1 μM) 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 α1B-1α2bβ1-2 Ca2+ channel subunits form channels in HEK.293 cells that exhibit properties consistent with the activity of the native N-type VDCC previously described in neurons.
- HEK293 cells
- fura-2 based microfluorimetry
ASJC Scopus subject areas
- Cellular and Molecular Neuroscience