Mucopolysaccharidose type I is a lysosomal storage disease caused by a deficiency in the enzyme α-l-iduronidase (IDUA). The existence of a secretory pathway for lysosomal enzymes and the capture of secreted molecules by distant cells through binding to mannose-6-phosphate receptors have provided a rationale for enzyme replacement therapy in lysosomal storage diseases. We have used genetically modified fibroblasts implanted into neo-organs as an in vivo delivery system for IDUA. The human IDUA cDNA was isolated and inserted into a retroviral vector where it was expressed from the phosphoglycerate kinase 1 gene promoter. MPS I fibroblasts transduced with this vector showed high levels of IDUA activity and secreted phosphory-lated molecules that could be internalized by naive deficient cells. Neo-organs containing 2 × 107 IDUA-secreting cells were implanted into nude mice. Human and murine IDUA activities were measured in the liver and spleen of animals sacrificed 35–77 days after implantation. Human IDUA activity corresponded to 0.6–2.3% of the murine enzyme activity in the liver and to 0.1–0.3% in the spleen. These data indicated that human IDUA was secreted from neo-organs and internalized in distant tissues. An approach for enzyme replacement in mucopolysaccharidose type I (MPS I) has been examined. Skin fibroblasts were transduced with a retroviral vector containing the human α-l-iduronidase cDNA and implanted as neo-organs in nude mice. The presence of the human enzyme in the liver and spleen was detected in treated animals. These data suggest that the implantation of engineered fibroblasts into neo-organs may be of a therapeutic benefit in MPS.
Abstract Two C‐terminal splice variants (BI‐1 and BI‐2, now termed Ca v 2.1a and Ca v 2.1b) of the neuronal voltage‐gated P/Q‐type Ca 2+ channel α 1A 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 Ba 2+ currents (I Ba ) 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 I Ba caused by coexpression of the β 4 auxiliary subunit were substantially different in BI‐1‐ and BI‐2‐containing channels in comparison to those induced by β 3 . 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 β 4 with the distinct α 1A isoforms. These interactions appear to favour the binding of β 4 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 α 1A 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 α 1A and β 4 are affected.
We have investigated the molecular mechanisms whereby the I-II loop controls voltage-dependent inactivation in P/Q calcium channels. We demonstrate that the I-II loop is localized in a central position to control calcium channel activity through the interaction with several cytoplasmic sequences; including the III-IV loop. Several experiments reveal the crucial role of the interaction between the I-II loop and the III-IV loop in channel inactivation. First, point mutations of two amino acid residues of the I-II loop of Cav2.1 (Arg-387 or Glu-388) facilitate voltage-dependent inactivation. Second, overexpression of the III-IV loop, or injection of a peptide derived from this loop, produces a similar inactivation behavior than the mutated channels. Third, the III-IV peptide has no effect on channels mutated in the I-II loop. Thus, both point mutations and overexpression of the III-IV loop appear to act similarly on inactivation, by competing off the native interaction between the I-II and the III-IV loops of Cav2.1. As they are known to affect inactivation, we also analyzed the effects of β subunits on these interactions. In experiments in which the β4 subunit is co-expressed, the III-IV peptide is no longer able to regulate channel inactivation. We conclude that (i) the contribution of the I-II loop to inactivation is partly mediated by an interaction with the III-IV loop and (ii) the β subunits partially control inactivation by modifying this interaction. These data provide novel insights into the mechanisms whereby the β subunit, the I-II loop, and the III-IV loop altogether can contribute to regulate inactivation in high voltage-activated calcium channels.
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, Cavalpha, with the auxiliary subunit, Cavbeta. In the absence of this auxiliary subunit, Cavalpha 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 CD8 alpha chimeras, the molecular determinants of Cavalpha2.1 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 Cavalpha2.1 contains multiple ER-retention determinants beside the beta 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 Cavalpha2.1, also produce ER retention. It is also not an obligatory determinant as, similarly to low-threshold calcium channels, the I-II loop of Cavalpha1.1 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 Cavalpha in order to mask several internal ER-retention determinants and (ii). the association of other proteins, including the Cavbeta subunit, to suppress the remaining ER-retention determinants.
Abstract Mammalian synaptotagmins constitute a multigene family of at least 11 membrane proteins. We have characterized synaptotagmin IV using antibodies directed against the C2A domain of the protein. Antibodies reacted specifically with a protein band that migrated as a 41–44 kDa doublet. Synaptotagmin IV expression was regulated throughout development. A strong decrease in the amount detected by Western blotting occurred between postnatal day 5 and adulthood, in agreement with studies on the expression of synaptotagmin IV transcripts. In subcellular fractionation, synaptotagmin IV was not detected in the synaptic vesicle‐enriched fraction. Immunofluorescence microscopy was concordant with this finding. In 6‐day‐old rat cerebellum and cultured hippocampal neurons the subcellular distribution of synaptotagmin IV was clearly different from that of synaptotagmin I. Synaptotagmin IV displayed a punctate non‐polarized distribution on neuronal extensions, whereas synaptotagmin I staining was essentially synaptic. Synaptotagmin IV staining was also observed in the soma in strong perinuclear fluorescent puncta superimposed on that of Golgi/TGN markers. Furthermore, synaptotagmin IV was seen in the proximal part of the growth cone domain and not in the microfilament‐rich region which includes filopodia. Co‐localizations with the adhesion molecules vinculin and zyxin at the proximal part of growth cones were observed. Synaptotagmin IV may thus be involved in the regulation of specific membrane‐trafficking pathways during brain development.
Spinocerebellar ataxia type 6 (SCA6) is a dominantly inherited neurodegenerative disease caused by a small expansion of CAG repeats in the sequence coding for the cytoplasmic C-terminal region of the Ca(v)2.1 subunit of P/Q-type calcium channels. We have tested the toxicity of mutated Ca(v)2.1 C-terminal domains expressed in the plasma membrane. In COS-7 cells, CD4-green fluorescent protein fused to Ca(v)2.1 C-terminal domains containing expanded 24 polyglutamine (Q) tracts displayed increased toxicity and stronger expression at the cell surface relative to 'normal' 12 Q tracts, partially because of reduced endocytosis. Glutathione S-transferase pull-down and proteomic analysis indicated that Ca(v)2.1 C-termini interact with the heavy and light chains of cerebellar myosin IIB, a molecular motor protein. This interaction was confirmed by coimmunoprecipitation from rat cerebellum and COS-7 cells and shown to be direct by binding of in vitro-translated (35)S-myosin IIB heavy chain. In COS-7 cells, incremented polyglutamine tract length increased the interaction with myosin IIB. Furthermore, the myosin II inhibitor blebbistatin reversed the effects of polyglutamine expansion on plasma membrane expression. Our findings suggest a key role of myosin IIB in promoting accumulation of mutant Ca(v)2.1Ct at the plasma membrane and suggest that this gain of function might contribute to the pathogenesis of SCA6.
The growth factor interleukin 2 (IL2) binds to and is internalized together with high-affinity surface receptors present on lymphoid cells. This endocytosis thus results in down-regulation of the receptors. However, it is not known if the internalization is relevant to the induction of cell growth. In the present study a rat monoclonal antibody to the P55 chain of the IL2 receptor was used to examine the role of receptor internalization in the IL2-dependent autocrine human tumor T cell line IARC 301. When given alone, this antibody did not inhibit IL2 binding, internalization, or IL2-dependent cell proliferation. However, crosslinking by anti-rat immunoglobulins, which did not affect binding of the growth factor, inhibited both IL2 internalization and cell proliferation. Besides offering a novel means for the specific inhibition of the uptake of IL2 bound to IL2 high-affinity receptors, the results are compatible with the association of this receptor-ligand uptake to the growth stimulation by IL2.
