Year: 2022
Type: dissertation
Abstract: B cell activation leads to the production of antibodies that protect us from infection. Recognition of antigen by B cells begins with the B cell receptor (BCR) recognizing and binding antigen, which initiates BCR signalling and antigen internalization. Calcium is an important secondary messenger during B cell activation and calcium signalling in B cells is best characterized by the store operated calcium entry (SOCE) pathway that triggers ion channels on the plasma membrane, predominantly known to be calcium release-activated calcium (CRAC) channels. Patients that have mutations in the proteins of this pathway present with severe immunodeficiency; however, murine B cells deficient in CRAC channel components mount normal antibody responses, suggesting that alternative channels may be important. We identified a member of the calcium permeable transient receptor potential (TRP) ion channel family, TRPV5, as a candidate channel expressed in B cells by a qPCR screen. We found TRPV5 polarized to BCR clusters upon stimulation in a RhoA dependent manner and siRNA knockdown of TRPV5 in a murine B cell line impaired calcium flux upon BCR stimulation. To further investigate the role of TRPV5 in B cell responses, we generated a murine TRPV5 knockout (KO) by CRISPR-Cas9. TRPV5 KO mice had normal B cell development and mature B cell numbers. TRPV5 KO B cells had impaired spreading and contraction in response to membrane-bound antigen. Surprisingly, calcium influx upon BCR stimulation in primary TRPV5 KO B cells was not impacted; however, differential expression of other calcium-regulating proteins, such as ORAI1, which we found was expressed 5-fold higher in TRPV5 KO B cells than WT B cells, may contribute to a compensatory mechanism. Despite no difference in calcium signalling, BCR signalling measured by phospho-tyrosine residues was reduced in TRPV5 KO B cells. Consistent with this, immunization induced T-dependent (TD) antigen-specific antibody titers were reduced in TRPV5 KO mice compared to WT mice. Thus, our findings identify a role for TRPV5 in BCR signalling and B cell activation.
Source: TSpace (University of Toronto)
Author: Mahtani, Trisha
Institution: University of Toronto
Language: English
Topic: Ion Channels and Receptors
Subfield: Sensory Systems
Field: Neuroscience
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7132907841