title: Role of ion channels in controlling murine corticotrophin excitability
Creators: name: Nair, Sooraj Venugopalan
Publisher: name: The University of Edinburgh
Year: 2023
Subjects: subject: corticotrophs
subject: pseudoplateau bursting
subject: ion channels
subject: dexamethasone

description: Anterior pituitary corticotrophs are integral components of the hypothalamus-pituitary-adrenal (HPA) axis controlling the neuroendocrine stress response. A variety of ion channels regulate intrinsic electrical excitability of corticotrophs and are also targets for control of excitability by the hypothalamic neuropeptides CRH/AVP as well as negative feedback by glucocorticoids. The context under which ion channels are regulated allows corticotrophs to transition between excitability patterns including from single spikes to ‘pseudoplateau bursting’. Hence, ion channels are regulators of feed forward mechanisms of ACTH secretion and feedback mechanisms of glucocorticoids (GCs) regulating the HPA axis function at the pituitary level. However, a comprehensive analysis of the expression of various ion channels has not yet been determined in corticotrophs. Furthermore, the functional role of different ion channels is poorly understood and whether ion channel expression is changed under different conditions, including during chronic stress or is different between sexes is unknown. In Chapter 3, analysis of ion channel mRNA expression by RNA-seq from corticotrophs FACS purified from POMC-GFP mice was undertaken to determine the landscape of ion channel expression and to determine whether this was different between males and females or was modified in response to chronic stress or long term glucocorticoid exposure. Corticotrophs express a wide range of ion channels however differential expression between sexes points to the possibility of changes in corticotroph excitability between sexes. In males, exposure to 2 weeks of daily restraint stress resulted in modest changes in ion channel gene expression however, following 4 weeks after recovery a number of ion channel mRNAs were significantly downregulated. In males treated for 4 weeks with dexamethasone in the drinking water relatively small changes in ion channel expression were observed after treatment or up to 4 weeks after dexamethasone withdrawal. Indeed, few ion channels were differentially regulated by both chronic stress and dexamethasone exposure although members of the Kv 12.x, Trp and Ttyh ion channel families were differentially expressed in both datasets. In Chapter 4, the functional role of Kcnh2 (Kv 11.1) one of the most highly expressed ion channel mRNAs in our FACS-purified male and female corticotrophs was determined by patch clamp electrophysiology. In several pituitary cell types, members of ether-a-go-go related (ERG, Kv 11.x) family of voltage dependent potassium channels are reported to control both spontaneous and hormone-induced regulation of excitability; however, their role in corticotrophs is not known. Erg-like currents were biophysically and pharmacologically isolated in corticotrophs from both sexes using E4031 (5 µM), a selective inhibitor of ERG family. Both male and female corticotrophs displayed variable Erg-like current densities with a V50 that would suggest they are active during spontaneous activity in both sexes. E4031 did not alter spontaneous electrical activity in either sex, however, CRH/AVP evoked excitability was inhibited by E4031. In particular, CRH-induced bursting in males and CRH/AVP evoked event frequency in both males and females was reduced by E4031 as well as by another Erg family inhibitor, dofetilide (DOF). However, CRH/AVP did not directly regulate erg-like currents in males or females in voltage clamp studies. This indicates ERG family plays a role in evoked corticotroph excitability, however Erg-ike currents are not a direct target for CRH/AVP. In Chapter 5, characterisation of the NMDG+ sensitive background sodium conductance previously identified in female corticotrophs was examined. The background current was characterised at the potassium reversal potential (EK) and NMDG+ decreased the background currents while external low calcium increased the background currents. While CRH/AVP failed to depolarise corticotrophs in the presence of NMDG+ , CRH/AVP had no significant effect on the background sodium current at EK suggesting it is not a direct target for regulation. NALCN, previously reported to encode background sodium currents in a number of cell types was one of the highly expressed ion channel mRNAs from our RNA seq analysis. While the background sodium current in corticotrophs displayed characteristics of NALCN more studies need to be undertaken to identity if they are responsible. In conclusion, murine corticotrophs exhibit differential expression of ion channel mRNAs between sexes, which may explain the differences in their intrinsic excitability. Gene expression of ion channel changes in response to chronic stress and long-term dexamethasone treatment seems evident, however the functional significance remains to be examined. ERG family plays an indirect role in evoked corticotroph excitability, however their role in CRH-induced bursting activity needs to be further explored. The background NMDG+ sensitive sodium conductances are important for electrical excitability and are sensitive to low extracellular calcium but do not appear to be direct targets for CRH/AVP. However, whether NALCN channels directly contribute to these needs are to be further explored. Thus, this thesis has revealed that challenge to HPA axis can change the expression of ion channels in corticotrophs and that ERG family potassium channels are an important determinant of corticotroph excitability.