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.

Human neuronal voltage-dependent calcium channels: studies on subunit structure and role in channel assembly.

Voltage-dependent calcium (Ca2+) channels, expressed in the CNS, appear to be multimeric complexes comprised of at least alpha 1, alpha 2 and beta subunits. Previously, we cloned and expressed human neuronal alpha 1, alpha 2 and beta subunits to study recombinant channel complexes that display properties of those expressed in vivo. The alpha 1B-mediated channel subtype binds omega-conotoxin (CgTx) GVIA with high affinity and exhibits properties of N-type voltage-dependent Ca2+ channels. Here we describe several alpha 2 and beta splice variants and report results on the expression of omega-CgTx GVIA binding sites, assembly of the subunit complex and biophysical function of alpha 1B-mediated channel complexes containing some of these splice variants. We optimized recombinant expression in human embryonic kidney (HEK) 293 cells of alpha 1B alpha 2b beta 1 subunit complexes by controlling the expression levels of subunit mRNAs and monitored cell surface expression by binding of omega-CgTx GVIA to the alpha 1B subunit. Co-expression of either alpha 2b or beta 1 subunits with an alpha 1B subunit increased expression of binding sites while the most efficient expression was achieved when both alpha 2b and beta 1 subunits were co-expressed with an alpha 1B subunit. The presence of alpha 2b affects the affinity of omega-CgTx GVIA binding and barium (Ba2+) current magnitudes, although it does not appear to alter kinetic properties of the Ba2+ current. This is the first evidence of an alpha 2 subunit modulating the binding affinity of a cell-surface Ca2+ channel ligand. Our results demonstrate that alpha 1, alpha 2 and beta subunits together contribute to the efficient assembly and functional expression of voltage-dependent Ca2+ channel complexes.

Developmental changes in calcium conductances contribute to the physiological maturation of cerebellar Purkinje neurons in culture.

The relationship between calcium conductances and developmental changes in the active and passive membrane properties of cerebellar Purkinje neurons from rats was studied in a culture model system by using current-clamp and voltage-clamp techniques. These cultures, at 6-21 d of age, represented the main period of morphological and physiological development of the Purkinje neuron. In the current-clamp studies, input resistance decreased and the current-voltage curve became more S-shaped as the neurons matured in culture. Spike-generating properties also changed. Immature Purkinje neurons without dendritic structure produced repetitive, fast TTX-sensitive simple spikes when stimulated electrically. The simple spike frequency increased with maturation. In older neurons (greater than or equal to 12 d in vitro) with well-developed dendritic structure, a burst event, the complex spike, preceded the repetitive simple spike firing. Magnesium (10 mM) and cadmium (50-100 microM), calcium channel blockers, antagonized the repetitive simple spike firing in both young and old neurons. The complex spike of the older neurons was also antagonized by magnesium (10 mM) but was resistant to cadmium (50-100 microM), suggesting that a pharmacologically distinct calcium conductance mediated this spike event. Whole-cell voltage-clamp recordings showed that the older Purkinje neurons expressed two calcium currents, a low-threshold rapidly inactivating calcium current resistant to cadmium (50-100 microM) and a high-threshold slowly inactivating calcium current antagonized by cadmium (50-100 microM). In young Purkinje neurons without dendritic structure (6-9 d in vitro), only the high-threshold calcium current was evident. The amplitude of this current increased approximately 50% during development. These results indicate that the developmental expression of calcium conductances plays a prominent role in the physiological maturation of the cultured Purkinje neurons, which closely simulate the physiologic cells they model. The high-threshold calcium conductance is expressed early in development and contributes to repetitive simple spike firing of both the young and old neurons. The low-threshold calcium conductance appears later in development, coincident with dendritic expression, and plays a major role in the generation of the complex spike.

Structure and functional characterization of neuronal alpha 1E calcium channel subtypes.

We have cloned overlapping cDNAs encoding alpha 1E Ca2+ channel subunits from mouse and human brain. We observed that these alpha 1E transcripts were widely distributed in the central nervous system. We also demonstrated the existence of two variants of the human alpha 1E subunit. Comparison of the sequence of these alpha 1E subunits to those from other species suggests that at least four alternatively spliced variants of alpha 1E exist. Expression of human alpha 1E in HEK293 cells and Xenopus oocytes produced high voltage-activated Ca2+ currents that inactivated rapidly (tau approximately 20 ms at 0 mV). The size of the currents obtained were enhanced approximately 40-fold by co-expression with human neuronal alpha 2 and beta Ca2+ channel subunits. alpha 1E currents were insensitive to the drugs and toxins previously used to define other classes of voltage-activated Ca2+ channels. Thus, alpha 1E-mediated Ca2+ channels appear to be a pharmacologically distinct class of voltage-activated Ca2+ channels.

The human N-methyl-D-aspartate receptor 2C subunit: genomic analysis, distribution in human brain, and functional expression.

cDNAs encoding four isoforms of the human NMDA receptor (NMDAR) NMDAR2C (hNR2C-1, -2, -3, and -4) have been isolated and characterized. The overall identity of the deduced amino acid sequences of human and rat NR2C-1 is 89.0%. The sequences of the rat and human carboxyl termini (Gly925-Val1,236) are encoded by different exons and are only 71.5% homologous. In situ hybridization in human brain revealed the expression of the NR2C mRNA in the pontine reticular formation and lack of expression in substantia nigra pars compacta in contrast to the distribution pattern observed previously in rodent brain. The pharmacological properties of hNR1A/2C were determined by measuring agonist-induced inward currents in Xenopus oocytes and compared with those of other human NMDAR subtypes. Glycine, glutamate, and NMDA each discriminated between hNR1A/2C-1 and at least one of hNR1A/2A, hNR1A/2B, or hNR1A/2D subtypes. Among the antagonists tested, CGS 19755 did not significantly discriminate between any of the four subtypes, whereas 5,7-dichlorokynurenic acid distinguished between hNR1A/2C and hNR1A/2D. Immunoblot analysis of membranes isolated from HEK293 cells transiently transfected with cDNAs encoding hNR1A and each of the four NR2C isoforms indicated the formation of heteromeric complexes between hNR1A and all four hNR2C isoforms. HEK293 cells expressing hNR1A/ 2C-3 or hNR1A/2C-4 did not display agonist responses. In contrast, we observed an agonist-induced elevation of intracellular free calcium and whole-cell currents in cells expressing hNR1A/2C-1 or hNR1A/2C-2. There were no detectable differences in the macroscopic biophysical properties of hNR1A/2C-1 or hNR1A/2C-2.