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 (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 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 mu M) and omega-conotoxin-MVITA (mu-CmTx-MVIIA; 100 nM-1 mu M) 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 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.