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

The human α_1B-1β_1-2Ca²⁺ 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 (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 CaCl₂. I_Ca 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 α_1B-1α_2bβ_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 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 I_Ca 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 α_1B-1α_2bβ_1-2 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.