Abstract by Feng Xue

Ion channels are membrane proteins that play critical role for ion homeostasis and communication between cells. Ion channel expression and function has to be tightly regulated, thus it is highly important to identify and study modulators of ion channels, such as protein-protein interactions and small molecule drugs. In this thesis two types of ion channels, Acid-sensing ion channels (ASICs) and voltage-sensitive sodium channels (Na_vs) are studied.

ASICs are a family of proton-gated ion channels expressed in the central and peripheral nervous system. They play an important role in physiological processes like synaptic plasticity, but also pathological processes, including tumor progressing and necroptosis in ischemic stroke. In this thesis, two protein families were found to directly interact with ASICs.

Previous mass spectrometry (MS) work suggested one group of ASIC interaction partners to be G protein α subunits (Gα). Gα proteins are activated in response to activation of G protein coupled receptors, and subsequently regulate to downstream signaling pathways. Previous research suggested for cellular pathways involving G proteins to regulate the surface expression of ASICs via its intracellular N- and C-terminal ends, but the exact mechanism remained unclear. In this thesis, I used pulldown assays to demonstrate a direct interaction between a subtype of ASICs, ASIC1a, and different members of the Gα family. Furthermore, by using truncations of ASIC1a, I found that Gα proteins can still interact directly with ASIC1a missing its intracellular C- and N termini. Moreover, using two-electrode voltage-clamp electrophysiology and surface expression assays, Gα was found to regulate the proton induced maximum currents of ASIC1a by regulating membrane surface expression. Importantly, this regulation does not appear to be dependent on cAMP-dependent pathways. Together, our work sheds new light on how ASIC function can be modulated by G proteins.

The other group of ASIC interaction partners identified by MS belong to the family of monocarboxylate transporters (MCTs), which play important roles in regulating intra and extracellular pH, as well as energy metabolism and metabolic homeostasis. Furthermore, MCTs are upregulated in cancer cells to support the high energy demand of these highly proliferating cells. A previous study suggested the potential interplay between ASIC1a and MCT in primary hippocampal and cortical neuron. To this end, we have recently found that MCTs modulate ASIC1a function in mammalian cells. In this thesis, a direct interaction between several ASIC subtypes and MCT family members was demonstrated using pulldown strategies. ASIC1a missing both intracellular N- and C-terminal tails did not diminish the interaction with MCT1, and two truncated MCT constructs, dividing the MCT into a N-terminal and C-terminal domain, were both able to interact with ASIC1a. Thus, these results indicates a rather large interaction surface between several transmembrane helices. Understanding the interaction surface between ASIC and MCTs might in the future pave the way for drugs targeting the interaction between ASICs and MCTs.

In a separate project, I applied structure-based in silico screening approaches to repurpose FDA approved drugs for Nav channel regulation, that targets an extracellular cavity of voltage-sensing domain IV (VSD_IV) of Na_v1.7. Nine compounds that displayed a high interaction score in the virtual screening and good solubility were selected as the hits for further in vitro testing. Using electrophysiological approaches, I found that application of 100 μM argatroban, labetalol, dabigatran or chenodeoxycholic acid reduced endogenous Na_v current amplitude in SH-SY5Y cells by more than 25%. The subtype selectivity, potency and dose-dependence of these compounds will be further tested in the future. In general, repurposing FDA-approved drugs for Nav channel related disease has the potential to accelerate the progress of clinical trials of these drugs. Also, finding potential off target effects of these drug against ion channels that are key to cellular excitability will be informative from a safety point of view.

In summary, two different ASIC protein-protein interactions were studied in this thesis. Gα regulates surface expression of ASIC1a in a cAMP-independent manner, while MCTs regulate the function of ASICs via a rather large, but direct interaction surface. Finally, four FDA approved drugs were repurposed to be potential Na_v channel inhibitor.