Validation of the suitability of mouse neuroblastoma cells in the study of chloride homeostasis using patch-clamp
1University of Oulu, Faculty of Science, Physics
|Online Access:||PDF Full Text (PDF, 2.1 MB)|
|Persistent link:|| http://urn.fi/URN:NBN:fi:oulu-202306292787
Oulu : A.-S. Mäkitiura,
|Publish Date:|| 2023-06-30
|Thesis type:||Master's thesis
The function of the nervous system is dependent on the balance between excitatory and inhibitory activity. In the central nervous system (CNS), the homeostasis of Cl- is in a key role in modulating neuronal excitability, since the intracellular Cl- concentration ([Cl-]i) determines the postsynaptic responses mediated by GABA and glycine, the main inhibitory neurotransmitters. Deficiency in the regulation of Cl- homeostasis, that is maintained by various ion channels and transporters, is associated with many neurological disorders such as epilepsy. Hence, the understanding of the mechanisms underlying the regulation of Cl- homeostasis is important.
Patch-clamp is an electrophysiological method that is used to study the electrical properties of excitable cells. It is a versatile technique, as it provides information of the concentration gradients of ions as well as ion channel kinetics. One important electrophysiological parameter describing channel function is the reversal potential of an ion (E(i)). Patch-clamp experiments can be done by using cultured cells or tissue slices, which can be genetically modified to express exogenous proteins, such as membrane transporters. There are a variety of cell lines that can be used as expression systems in patch-clamp, such as the commercial mouse neuroblastoma (N2a) cell line, that is used in the experiments in this thesis.
The aim of this thesis is to validate the suitability of the N2a cell line as an expression system for E(Cl) recordings using patch-clamp. In the experiments, whole-cell configuration of the patch-clamp was used to study the endogenous voltage-gated currents that are expressed in the N2a cell line, especially in the voltage-range wherein the E(Cl) typically lies.
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