Delphine Bichet

The I-II Loop of the Ca2+ Channel α1 Subunit Contains an Endoplasmic Reticulum Retention Signal Antagonized by the β Subunit

Abstract The auxiliary β subunit is essential for functional expression of high voltage-activated Ca 2+ channels. This effect is partly mediated by a facilitation of the intracellular trafficking of α 1 subunit toward the plasma membrane. Here, we demonstrate that the I-II loop of the α 1 subunit contains an endoplasmic reticulum (ER) retention signal that severely restricts the plasma membrane incorporation of α 1 subunit. Coimmunolabeling reveals that the I-II loop restricts expression of a chimera CD8-I-II protein to the ER. The β subunit reverses the inhibition imposed by the retention signal. Extensive deletion of this retention signal in full-length α 1 subunit facilitates the cell surface expression of the channel in the absence of β subunit. Our data suggest that the β subunit favors Ca 2+ channel plasma membrane expression by inhibiting an expression brake contained in β-binding α 1 sequences.

Coding and Noncoding Variation of the Human Calcium-Channel β4-Subunit Gene CACNB4 in Patients with Idiopathic Generalized Epilepsy and Episodic Ataxia

Inactivation of the beta4 subunit of the calcium channel in the mouse neurological mutant lethargic results in a complex neurological disorder that includes absence epilepsy and ataxia. To determine the role of the calcium-channel beta4-subunit gene CACNB4 on chromosome 2q22-23 in related human disorders, we screened for mutations in small pedigrees with familial epilepsy and ataxia. The premature-termination mutation R482X was identified in a patient with juvenile myoclonic epilepsy. The R482X protein lacks the 38 C-terminal amino acids containing part of an interaction domain for the alpha1 subunit. The missense mutation C104F was identified both in a German family with generalized epilepsy and praxis-induced seizures and in a French Canadian family with episodic ataxia. These coding mutations were not detected in 255 unaffected control individuals (510 chromosomes), and they may be considered candidate disease mutations. The results of functional tests of the truncated protein R482X in Xenopus laevis oocytes demonstrated a small decrease in the fast time constant for inactivation of the cotransfected alpha1 subunit. Further studies will be required to evaluate the in vivo consequences of these mutations. We also describe eight noncoding single-nucleotide substitutions, two of which are present at polymorphic frequency, and a previously unrecognized first intron of CACNB4 that interrupts exon 1 at codon 21.

Chemical synthesis and characterization of maurocalcine, a scorpion toxin that activates Ca2+ release channel/ryanodine receptors.

Maurocalcine is a novel toxin isolated from the venom of the chactid scorpion Scorpio maurus palmatus. It is a 33‐mer basic peptide cross‐linked by three disulfide bridges, which shares 82% sequence identity with imperatoxin A, a scorpion toxin from the venom of Pandinus imperator. Maurocalcine is peculiar in terms of structural properties since it does not possess any consensus motif reported so far in other scorpion toxins. Due to its low concentration in venom (0.5% of the proteins), maurocalcine was chemically synthesized by means of an optimized solid‐phase method, and purified after folding/oxidation by using both C18 reversed‐phase and ion exchange high‐pressure liquid chromatographies. The synthetic product (sMCa) was characterized. The half‐cystine pairing pattern of sMCa was identified by enzyme‐based cleavage and Edman sequencing. The pairings were Cys3‐Cys17, Cys10‐Cys21, and Cys16‐Cys32. In vivo, the sMCa was lethal to mice following intracerebroventricular inoculation (LD50, 20 μg/mouse). In vitro, electrophysiological experiments based on recordings of single channels incorporated into planar lipid bilayers showed that sMCa potently and reversibly modifies channel gating behavior of the type 1 ryanodine receptor by inducing prominent subconductance behavior.

Multiple determinants in voltage‐dependent P/Q calcium channels control their retention in the endoplasmic reticulum

Surface expression level of voltage‐dependent calcium channels is tightly controlled in neurons to avoid the resulting cell toxicity generally associated with excessive calcium entry. Cell surface expression of high voltage‐activated calcium channels requires the association of the pore‐forming subunit, Cavα, with the auxiliary subunit, Cavβ. In the absence of this auxiliary subunit, Cavα is retained in the endoplasmic reticulum (ER) through mechanisms that are still poorly understood. Here, we have investigated, by a quantitative method based on the use of CD8α chimeras, the molecular determinants of Cavα2.1 that are responsible for the retention, in the absence of auxiliary subunits, of P/Q calcium channels in the ER (referred to here as ‘ER retention’). This study demonstrates that the I–II loop of Cavα2.1 contains multiple ER‐retention determinants beside the β subunit association domain. In addition, the I–II loop is not the sole domain of calcium channel retention as two regions identified for their ability to interact with the I–II loop, the N‐ and C‐termini of Cavα2.1, also produce ER retention. It is also not an obligatory determinant as, similarly to low‐threshold calcium channels, the I–II loop of Cavα1.1 does not produce ER retention in COS7 cells. The data presented here suggests that ER retention is suppressed by sequential molecular events that include: (i) a correct folding of Cavα in order to mask several internal ER‐retention determinants and (ii) the association of other proteins, including the Cavβ subunit, to suppress the remaining ER‐retention determinants.

Distinct properties and differential β subunit regulation of two C‐terminal isoforms of the P/Q‐type Ca2+‐channel α1A subunit

Two C‐terminal splice variants (BI‐1 and BI‐2, now termed Cav2.1a and Cav2.1b) of the neuronal voltage‐gated P/Q‐type Ca2+ channel α1A pore‐forming subunit have been cloned (Mori et al., 1991, Nature, 350, 398–402). BI‐1 and BI‐2 code for proteins of 2273 and 2424 amino acids, respectively, and differ only by their extreme carboxyl‐termini sequences. Here, we show that, in Xenopus oocytes, the two isoforms direct the expression of channels with different properties. Electrophysiological analysis showed that BI‐1 and BI‐2 have peak Ba2+ currents (IBa) at a potential of +30 and +20 mV, respectively. The different C‐terminal sequence (amino acids 2229–2273) of BI‐1 caused a shift in steady‐state inactivation by +10 mV and decreased the proportion of fast component of current inactivation twofold. Likewise, the biophysical changes in IBa caused by coexpression of the β4 auxiliary subunit were substantially different in BI‐1‐ and BI‐2‐containing channels in comparison to those induced by β3. Several of these differences in β regulation were abolished by deleting the carboxyl‐terminal splicing region. By creating a series of GST fusion proteins, we identified two locations in the C‐terminal (Leu2090–Gly2229 for BI‐1 and BI‐2, and Arg2230–Pro2424 for BI‐2 only) that determine the differential interaction of β4 with the distinct α1A isoforms. These interactions appear to favour the binding of β4 to the AID site, and also the plasma membrane expression of BI‐2. These results demonstrate that the final segment of the C‐terminal affects α1A channel gating, interaction and regulation with/by the β subunits. The data will have several implications for the understanding of the biophysical effects of many channelopathies in which the carboxyl‐termini of α1A and β4 are affected.

Antibodies against the β subunit of voltage-dependent calcium channels in Lambert–Eaton Myasthenic Syndrome

Abstract Lambert–Eaton myasthenic syndrome is an autoimmune disease that impairs neuromuscular transmission. Several studies suggest that neurotransmitter release is reduced by an immune response directed against the calcium channel complex of nerve terminals. The immunoglobulin G fractions from Lambert–Eaton myasthenic syndrome patients immunoprecipitate solubilized neuronal N- and P/Q-type channels and in certain cases brain, skeletal and cardiac muscle L-type channels [El Far O. et al . (1995) J. Neurochem . 64, 1696–1702; Lennon V. A. and Lambert E. H. (1989) Mayo Clin. Proc . 64, 1498–1504; Sher E. et al . (1989) Lancet ii, 640–643; Suenaga A. et al . (1996) Muscle Nerve 19, 1166–1168]. These channel immunoprecipitation assays are considered as useful for the diagnosis of this syndrome. In this study, we demonstrate that two predominant neuronal voltage-dependent calcium channel β subunits ( β 3 and β 4 , of mol. wt 58 000) are general targets of Lambert–Eaton myasthenic syndrome autoantibodies. Of 20 disease sera tested, 55% were able to immunoprecipitate 35 S-labeled β subunits. All five patients affected with small-cell lung carcinoma were positive for the β -subunit immunoprecipitation assay. Interestingly, only a fraction of the β -subunit-positive sera was also able to immunoprecipitate N- and P/Q-type channels, suggesting that several of the β -subunit epitopes are masked in native channels. In accordance with this observation, we found that several β -positive sera were able to prevent the interaction between calcium channel α 1 and β subunits in vitro . In cases where sera were able to immunoprecipitate β subunits, N- and P/Q-type channels, the immunoprecipitation of both channel types was either partially or entirely mediated by β -subunit antibodies. Our results suggest that assays based on the immunoprecipitation of β subunits can be used as an additional test to assist in the diagnosis of Lambert–Eaton myasthenic syndrome.