Raghib, Ayesha; (2002) Expression, localisation and function of truncated forms of the voltage-dependent calcium channel alpha1b. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Voltage gated calcium channels (VDCC) exist minimally as a complex containing an a1 subunit and at least two accessory subunits β and α2-δ1. The α1 subunit forms the pore of the channel, and consists of four homologous domains connected by intracellular loops. Several mutations in the α1 subunit of VDCCs have been identified that predict the generation of truncated proteins. This study focused on the expression and localisation of truncated β1B subunits. Constructs containing domain I, domain I-II and domain III-IV of β1B were constructed using standard molecular biology techniques, and their subcellular localisation was studied using antibody to an intracellular epitope of β1B and an extracellular epitope of α2-δ1, which was assumed to form a complex with the Domain I-II and Domain III-IV. The constructs were also tagged with GFP or its spectral variants (XFP), in order to permit direct visualisation of the proteins. All the truncated constructs were expressed successfully in Cos-7 cells. Using laser scanning confocal microscopy, it was shown that both the untagged and XFP-tagged Domain I-II were plasma membrane localised in the absence and presence of accessory subunits, although the presence of β subunits appeared to increase plasma membrane localisation. However, the XFP-Domain I-II construct showed pronounced ER retention, which was not evident when the construct was visualised using antibodies. The subcellular localisation of untagged Domain III-IV also varied when compared to the tagged Domain III-IV ECFP construct. Antibodies to untagged Domain III-IV and the Domain III- IV/a2-δ1 complex revealed plasma membrane and cytoplasmic localisation. Plasma membrane localisation was not observed in the absence of accessory subunits. In contrast, Domain III-IV ECFP showed pronounced ER retention and no conclusive plasma membrane localisation, both in the absence and presence of accessory subunits. Thus, it appeared that the addition of the XFP-tag was altering the subcellular localisation of the truncated proteins. Coexpression of domains I-II and domains III-IV in the absence and presence of accessory subunits showed no detectable increase in plasma membrane localisation, although this combination did form functional channels. The XFP-tagged Domain I construct showed cytoplasmic and ER localisation, and was not detectable at the plasma membrane. The localisation of the untagged Domain I could not be investigated due to lack of antibodies. Additional studies were also performed to investigate the effect of the truncated proteins on the expression of GFP-tagged β1B. Coexpression of GFP-β1B with untagged Domain I and Domain I-II dramatically reduced the expression of GFP-β1B, as evidenced by the absence of fluorescence from GFP-β1B. In contrast, untagged Domain III-IV trapped GFP-β1B in the ER. These results indicate that truncated proteins of β1B may have a suppresive effect on the full- length channel, suggesting a possible role in the modulation of β1B.

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