Thesis (Ph.D.) - University of Queensland, 2004. / Includes bibliography.

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Activation of a5??1 integrin potentiates L-type calcium current in vascular smooth muscle, which is partly mediated by tyrosine phoshorylation of the a1c channel subunit. Expressed rabbit VSM and neuronal isoforms are also potentiated by a5??1 integrin activation and require dual phosphorylation of a1c by PKA and c-Src. To explore common mechanisms of regulation by a5??1 integrin, whole cell patch clamp experiments were used to investigate the effects of a5??1 integrin antibody on expressed cardiac calcium channels. In HEK cells transfected with a1c, ??2a and a2-d1 subunits alone, currents increased 1.8 ?? 2.0 fold on application of a5??1 antibody. The potentiation was almost completely abolished on the application of PKI, a highly specific Protein Kinase A (PKA) inhibitor. The expressed currents increased 2.0 ?? 2.2 fold on application of PKA activator 8-Br-cAMP, and abolished by PKI. Our results suggest that regulation of L-type calcium channels by a5??1 integrin is a general mechanism shared by VSM, neuronal and cardiac channels. However, in the cardiac isoform, only PKA phosphorylation is involved.

The Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder of impaired neuromuscular transmission. It is associated with small cell lung carcinoma (SCLC) in 60% of patients. There is considerable evidence that the defect in LEMS is caused by autoantibodies to voltage-gated calcium channels (VGCCs) on the nerve terminal. Two VGCC subtypes were demonstrated in cultured neuronal cell lines by the technique of K⁺-induced ⁴⁵Ca²⁺ influx: one subtype was inhibited by dihydropyridines (DHPs), the other by ω-conotoxin (ωCgTx). These may correspond to L and N channels previously described for neuronal tissue. The presence of these VGCC subtypes was confirmed by radioligand binding studies using [3 Hj-PN200-11 0 and 125 I-d)ωCgTx respectively. Pooled LEMS IgG inhibited K⁺-induced Ca²⁺ flux by 40% in SKNSH (human neuroblastoma) cells, while control IgG had no effect. The same LEMS pool reduced the density of ¹²⁵I-ωCgTx binding sites in SKNSH and MAR5 (human SCLC) cells by 57% and 43% respectively. These results provide further evidence that LEMS antibodies cause a loss of functional VGCCs. 78 LEMS and 88 control sera were tested for anti-VGCC antibodies by the precipitation of ¹²⁵I-ωCgTx-labelled VGCCs extracted from SKNSH cells. 42% of LEMS sera had significant levels of antibody (30-1466pM) compared to the healthy controls (<31pM). There was a high correlation between these results and those obtained using antigen extracted from MAR5 cells. Raised antibody titres (30-82pM) were also found among SCLC patients (47%) and patients with rheumatoid arthritis or systemic lupus erythematosus (56%). The incidence of positive sera was not significant among patients with other neurological disorders, including myasthenia gravis. Antibody titre did not correlate with disease severity across individuals. However, longitudinal studies in two LEMS patients showed an inverse relation between antibody titre and an electromyographic index of disease severity. Some of the antibodies detected may, therefore, be implicated in the neurological symptoms of LEMS. The assay may be a useful aid for the diagnosis of LEMS in some patients.

Thesis (Ph. D.)--University of Chicago, Dept. of Organismal Biology and Anatomy, August 2000. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph.D.)--University of Delaware, 2005. / Principal faculty advisor: Mary C. Farach-Carson, Dept. of Biological Sciences. Includes bibliographical references.