Characteristics of a human N-type calcium channel expressed in HEK293 cells

The human α 1 B−1 β 1−2 Ca 2+ 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 (I Ca) 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 CaCl 2. I Ca and associated rises in intracellular free calcium concentrations ([Ca 2+] i) were sensitive to changes in both the [Ca 2+] o and holding potential. Steady-state inactivation was half maximal at a holding potential of — 60 mV. Ba 2+ was a more effective charge carrier than Ca 2+ through the α 1 B−1 α 2 b β 1−2 Ca 2+ channel and combinations of both Ba 2+ and Ca 2+ as charge carriers resulted in the anomalous mole fraction effect. Ca 2+ 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 Ca 2+ channels. The inorganic ions, gadolinium (Gd 3+), cadmium (Cd 2+) and nickel (Ni 2+) reduced the I Ca under voltage-clamp conditions in a concentration-dependent manner. The order of potency of the three ions was Gd 3+,Cd 2+,Ni 2+. These experiments suggest that the cloned and expressed α 1 B−1 α 2 b β 1−2 Ca 2+ 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.