Native N-type Research Articles

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.

Identification of a syntaxin-binding site on N-Type calcium channels

Immunochemical studies have suggested a tight association of syntaxin with N-type calcium channels. Syntaxin specifically interacts with the fusion proteins containing the cytoplasmic loop (LII-III) between homologous repeats II and III of the alpha 1 subunit of the class B N-type calcium channel (alpha 1B) from rat brain, but not with those of the class A Q-type (alpha 1A) or the class S L-type (alpha 1S) calcium channels. This interaction is mediated by an 87 amino acid sequence (773-859) containing two overlapping predicted helix-loop-helix domains. The 87 amino acid peptide can specifically block binding of native N-type calcium channels to syntaxin, indicating that this binding site is required for stable interaction of these two proteins. Interaction takes place with the C-terminal one-third of syntaxin (residues 181-288), which is thought to be anchored in the presynaptic plasma membrane. Our results suggest a direct interaction between the cytoplasmic domains of these two presynaptic membrane proteins that could have an important role in the targeting and docking of synaptic vesicles near N-type calcium channels, enabling tight structural and functional association of calcium entry sites and neurotransmitter release sites.

Functional properties of the purified N-type Ca2+ channel from rabbit brain.

N-type Ca2+ channels control a variety of key neuronal functions including transmitter release at synaptic terminals. The purified omega-conotoxin receptor from rabbit brain is a multisubunit complex composed of alpha 1B, alpha 2 delta, beta 3, and 95-kDa subunits. Immunoadsorption experiments confirm that the purified preparation does not contain alpha 1 subunits other than the omega-conotoxin-sensitive class B isoform. The functional properties of the purified channel have been analyzed further in lipid bilayers, and similarities to or differences from the native N-type Ca2+ channel have been outlined. Conserved properties include ion selectivity, open-time duration, and pharmacology (insensitivity to drugs affecting skeletal muscle L-type Ca2+ channels). Observed properties of the reconstituted channel which differ from the native channel include (a) sustained channel activity without Ca(2+)- or voltage-induced inactivation; (b) examples of extremely high open-state probability; (c) the absence of "run-down"; and (d) voltage independence of the Ca2+ channel gating. In addition, the conductance of the purified receptor is comprised between 7 and 27 picosiemens. Our results suggest that cellular components may play critical roles in the regulation of several biophysical properties and neuronal function of the native N-type Ca2+ channel.