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Publication Detail
Visualisation of the Native N-type Calcium Channel
  • Publication Type:
    Thesis/Dissertation
  • Authors:
    Ramgoolam K
  • Date awarded:
    2020
  • Awarding institution:
    UCL (University College London)
  • Language:
    English
Abstract
N-type calcium channels (CaV2.2) are important for neurotransmitter release in the central and peripheral nervous system. Immunohistochemical detection of native CaV2.2 has not been possible until now due to the low expression of these channels and lack of suitable antibodies. The present study utilises the recently developed constitutive knock-in (KI) transgenic mouse, expressing CaV2.2 with an epitope tag (2 x haemagglutinin; HA) inserted in the extracellular loop between S3 and S4 of domain II (CaV2.2_HAKIKI). The tag does not affect the function of the channel when expressed in vitro (Cassidy et al., 2014). In the somatosensory nervous system, the data show that CaV2.2_HA is expressed on the cell surface of dorsal root ganglion (DRG) neurons. In the spinal cord, CaV2.2_HA is predominantly in the superficial laminae LIand LII of the dorsal horn, mainly in the primary afferent terminals, since there is a reduction in CaV2.2_HA staining following rhizotomy. Co-cultures between DRG and spinal cord neurons permit the study of CaV2.2_HA at the presynaptic terminal. Super-resolution images of the synapses formed by these co-cultures revealed the regulation of CaV2.2_HA expression at the presynaptic terminal over time in culture. Preliminary studies of TRPV1 activation by capsaicin on cell surface CaV2.2_HA was tested on cultured DRG neurons. Short incubations (20 s) with capsaicin increases CaV2.2_HA expression at the cell membrane of small, medium and large-diameter neurons. However, following longer incubations (2 to 4 min) with capsaicin, there is a substantial decrease of CaV2.2_HA immunoreactivity at the membrane. Nevertheless, further studies are required to determine whether this is mediated by a TRPV1-dependent or -independent mechanism. The CaV2.2_HAKIKI mice will be instrumental in future studies to enhance the understanding of the presynaptic role of endogenous N-type calcium channels in physiological and pathological states.
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