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

doi:10.1016/0028-3908(93)90004-M

Neuropharmacology

Volume 32, Issue 11, November 1993, Pages 1089-1095, 1097-1102

https://doi.org/10.1016/0028-3908(93)90004-M Get rights and content

Abstract

Voltage-dependent calcium (Ca²⁺) channels, expressed in the CNS, appear to be multimeric complexes comprised of at least α₁, α₂ and β subunits. Previously, we cloned and expressed human neuronal α₁, α₂ β subunits to study recombinant channel complexes that display properties of those expressed in vivo. The α_1B-mediated channel subtype binds ω-conotoxin (CgTx) GVIA with high affinity and exhibits properties of N-type voltage-dependent Ca²⁺ channels. Here we describe several α₂ and β splice variants and report results on the expression of ω-CgTx GVIA binding sites, assembly of the subunit complex and biophysical function of α_1B-mediated channel complexes containing some of these splice variants. We optimized recombinant expression in human embryonic kidney (HEK) 293 cells of α_1Bα_2bβ₁, subunit complexes by controlling the expression levels of subunit mRNAs and monitored cell surface expression by binding of ω-CgTx GVIA to the α_1B subunit. Co-expression of either α_2b or β₁ subunits with an α_1B subunit increased expression of binding sites while the most efficient expression was achieved when both α_2b and β₁ subunits were ωexpressed with an α_1B subunit. The presence of α_2b affects the affinity of ω-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 α₂ subunit modulating the binding affinity of a cell-surface Ca²⁺ channel ligand. Our results demonstrate that α₁, α₂ and β subunits together contribute to the efficient assembly and functional expression of voltage-dependent Ca²⁺ channel complexes.

References (41)

E. Bosse et al.
The cDNA and deduced amino acid sequence of the gamma subunit of the L-type calcium channel from rabbit skeletal muscle
FEBS Lett.
(1990)
K.P. Campbell et al.
The biochemistry and molecular biology of the dihydropyridine-sensitive calcium channel
Trends Neurosci.
(1988)
A. Castellano et al.
Cloning and expression of a third calcium channel β subunit
J. Biol. Chem.
(1993)
Y. Gu et al.
Identification of two amino acid residues in the ϵ subunit that promote mammalian muscle acetylcholine receptor assembly in COS cells
Neuron
(1991)
P. Lory et al.
The β subunit controls the gating and dihydropyridine sensitivity of the skeletal muscle Ca²⁺ channel
Biophys. J.
(1992)
P. Lory et al.
Characterization of β subunit modulation of a rabbit cardiac L-type Ca²⁺ channel α₁ subunit as expressed in mouse L cells
FEBS Lett.
(1993)
T. Niidome et al.
Molecular cloning and characterization of a novel calcium channel from rabbit brain
FEBS Lett.
(1992)
E. Perez-Reyes et al.
Cloning and expression of a cardiac/brain β subunit of the L-type calcium channel
J. Biol. Chem.
(1992)
M. Pragnell et al.
Cloning and tissue-specific expression of the brain calcium channel β-subunit
FEBS Lett.
(1991)
D. Singer-Lahat et al.
Evidence for the existence of RNA of Ca²⁺-channel $α_{2} δ$ subunit in Xenopus ooxytes
Biochim. Biophys. Acta
(1992)

T.P. Snutch et al.

Ca²⁺ channels: diversity of form and function

Curr. Opin. Neurobiol.

(1992)

M.E. Williams et al.

Structure and functional expression of α₁, α₂, and β subunits of a novel human neuronal calcium channel subtype

Neuron

(1992)

E. Bosse et al.

Stable and functional expression of the calcium channel α 1 subunit from smooth muscle in somatic cell lines

EMBO J.

(1992)

J. Boulter et al.

Purification of an acetylcholine receptor from a nonfusing muscle cell line

Biochemistry

(1977)

A. Castellano et al.

Cloning and expression of a fourth neuronal calcium channel beta subunit

Biophys. J.

(1993)

R.A. Chavez et al.

Subunit folding and αδ heterodimer formation in the assembly of the nicotinic acetylcholine receptor

J. Biol. Chem.

(1992)

N. Dascal et al.

Expression and modulation of voltage-gated calcium channels after RNA injection in Xenopus oocytes

Science

(1986)

S.B. Ellis et al.

Sequence and expression of mRNAs encoding the α₁ and α₂ subunits of a DHP-sensitive calcium channel

Science

(1988)

Y. Gu et al.

Assembly of the mammalian muscle acetylcholine receptor in transfected COS cells

J. Cell Biol.

(1991)

R. Hullin et al.

Calcium channel β subunit heterogeneity: functional expression of cloned cDNA from heart, aorta and brain

EMBO J.

(1992)

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

Abstract

FEBS Lett.

Trends Neurosci.

J. Biol. Chem.

Neuron

Biophys. J.

FEBS Lett.

FEBS Lett.

J. Biol. Chem.

FEBS Lett.

Biochim. Biophys. Acta

Curr. Opin. Neurobiol.

Neuron

Stable and functional expression of the calcium channel α 1 subunit from smooth muscle in somatic cell lines

EMBO J.

Purification of an acetylcholine receptor from a nonfusing muscle cell line

Biochemistry

Cloning and expression of a fourth neuronal calcium channel beta subunit

Biophys. J.

Subunit folding and αδ heterodimer formation in the assembly of the nicotinic acetylcholine receptor

J. Biol. Chem.

Expression and modulation of voltage-gated calcium channels after RNA injection in Xenopus oocytes

Science

Sequence and expression of mRNAs encoding the α1 and α2 subunits of a DHP-sensitive calcium channel

Science

Assembly of the mammalian muscle acetylcholine receptor in transfected COS cells

J. Cell Biol.

Calcium channel β subunit heterogeneity: functional expression of cloned cDNA from heart, aorta and brain

EMBO J.

Sequence and expression of mRNAs encoding the α₁ and α₂ subunits of a DHP-sensitive calcium channel