The Role of Leak Channels in Regulating Endocrine Pituitary Cell Excitability

Abstract

The pituitary gland produces a variety of hormones that regulate other glands and organs throughout the body to control critical bodily functions including growth, metabolism and the stress response. Endocrine pituitary cells are electrically excitable: they generate action potentials to regulate their intracellular calcium level ([Ca2+]i) and eventually hormone secretion. The interplay between hypothalamic neurohormones and feedback signals coming from peripheral endocrine glands controls hormone secretion from pituitary cells by regulating the properties of ion channels and in turn the pattern of electrical activity. Therefore, elucidating the mechanisms underlying hormone secretion involves characterisation of ionic conductances which govern pituitary cell excitability. This PhD thesis explores how sodium and potassium leak channels regulate pituitary cell excitability. Leak channels play an important role in tuning the resting membrane potential, appropriately maintaining it close to the threshold for generating action potentials in all pituitary cells. The dynamic clamp electrophysiology technique was used to virtually vary the conductance of the leak channels, and evaluate their effect on the pattern of electrical activity and intracellular calcium concentration in the GH4 lacto-somatotroph cell line as well as in murine primary pituitary cells. It was found that very small alterations in the conductance of sodium and potassium leak channels result in substantial changes in the patterns of electrical activity and intracellular calcium oscillations in both GH4 and primary pituitary cells. Increasing the conductance of sodium leak channels by only a few fractions of a nanosiemen (nS) enhanced the excitability of pituitary cells significantly. In contrast, increasing the conductance of potassium leak channels by comparable values reduced the excitability and intracellular calcium concentration of the cells. Despite the crucial role of sodium leak conductance in tuning the resting membrane potential at depolarised levels away from the potassium equilibrium potential, the molecular identity of this channel in pituitary cells has remained unknown. One candidate protein channel is the sodium leak channel, non-selective (NALCN). The NALCN channel is widely expressed in the central nervous system, and has been characterised as a key modulator of cell excitability in several neuronal populations. Hence, in the second stage of my PhD, we constructed a lentiviral vector to knock down the NALCN channel in murine primary anterior pituitary cells in culture, and evaluate NALCN’s role in regulating cell excitability and intracellular calcium concentration using electrophysiology and calcium imaging techniques. We discovered that: (1) NALCN encodes for sodium leak channel to acutely adjust the resting membrane potential and sustain intrinsically-regulated spontaneous firing in endocrine pituitary cells; (2) the NALCN channel is crucial for maintaining spontaneous [Ca2+]i oscillations; (3) NALCN mediates the major depolarising inward leak current in pituitary cells; and (4) as in neurons, extracellular calcium inhibits NALCN activity in pituitary cells. These discoveries advance our understanding of how cell excitability and consequently hormone secretion is regulated in endocrine pituitary cells.