Dissection of voltage-gated ion channel physiology in vivo in the C. elegans oxygen-sensing neural circuit

Neurons are excitable because of gradients of ions across their outer membrane. Ion channels in the membrane regulate the excitability of these nerve cells by selectively opening to let specific ions pass through. Our work focusses on a subfamily of ion channels that open or close depending on the cell's electrical state, and may cause epilepsy or schizophrenia when mutated in humans. When overexpressed they may turn normal cells into cancer cells, and it has been speculated that this is because the channels affect how cells respond to oxygen. They are also abundant in healthy brains but their function there is mysterious. I have found a way to shed light on their normal brain function by using a small nematode worm called Caenorhabditis elegans, a popular model organism in biology because it offers many powerful techniques giving results quickly. Worms contain a homologous (equivalent) gene that closely resembles the human channel. I studied mutants that lack the gene and found that they are defective in how these worms respond to the level of ambient oxygen, because their oxygen-sensing neurons don't function normally. We now propose to take advantage of these observations to elucidate the normal neural function of these channels. We will remove parts of the gene that code for individual parts of the protein and observe if the behaviour of the worm in response to oxygen changes, if the activity of the oxygen-sensing neurons is altered, or the localisation of the protein within the cell is different. We will also conduct a genetic search for other genes that interact with the channel or regulate it, by mutagenising animals and looking for progeny that behaves as if the channel is gone. In preliminary studies I have found one interaction partner that was not known before - a protein which is also involved in shaping how worms respond to oxygen. We aim to understand this interaction. Our proposal represents an opportunity to understand the function of these medically relevant ion channels.

Voltage-gated ion channels are highly conserved in evolution and broadly expressed in nervous systems. However, the neurophysiological function of many of these channel families remains mysterious, as their investigation in nervous systems in vivo has been hindered by a lack of tractable null mutant phenotypes. Voltage-activated channels have been linked to O2 sensing, but their mechanistic role in this is not understood.
I have identified several novel, distinct and genetically separable behavioural phenotypes of null mutants of a conserved ion channel in C. elegans, which alter the function of the [O2]-sensing neural circuit and thus oxygen responses, and discovered a novel interaction of the channel with genes that tune acute O2 responses. I will take advantage of these phenotypes to shed light on the mechanism of channel action by 1. performing a structure-function analysis of the channel and its domains; 2. investigating their functional interaction with other sensory proteins in tuning O2 responses; and 3. conducting an EMS mutagenesis screen, using aggregation behaviour as readout, to identify novel interactors / regulators of the channel. We will identify molecular lesions by whole-genome sequencing and characterising the interaction of 2-3 of the hits with the channel. Microfluidics chambers will be used to assay O2-evoked behaviour and neuronal Ca2+ with imaging of YC2.60; confocal microscopy of GFP-tagged channels will be used to assess aberrant localisation of truncated channels. We will complement our C. elegans studies with recording macropatch currents in HEK293 cells expressing the channel or a human homologue. This way, we aim to make significant contributions towards understanding the neurophysiology of O2-linked ion channels.

Scientists:
Due to the multidisciplinary nature of this project, we expect our results to find interest with multiple groups of scientists, by informing their own work, by advancing the understanding of O2-sensing mechanisms and ion channels, and by generating transgenic tools and strains available from us. In particular it will benefit neuroscientists, especially those studying ion channels and neural mechanisms of O2 sensing; it will also find particular interest in the community of C. elegans researchers and of cancer scientists, particularly those who study oxygen homeostasis in tumours. To maximise scientific benefit, we will disseminate our results widely by publication in high-profile journals and by presenting them at international and national meetings. Moreover, the proposed screen for novel interactors represents an excellent basis for our future research and its continued funding.

The postdoctoral researcher and technician working on this project will benefit from training in widely applicable state-of-the-art techniques in C. elegans neuroscience, and the exposure to the cutting-edge research of other Edinburgh neuroscientists. I will also benefit from training in in vitro electrophysiological recordings by our collaborators.

Our project will strengthen scientific collaborations within our Centre, the University and beyond. We will complement the range of phenomena we can investigate in C. elegans by adding in vitro electrophysiology to our repertoire. We will also deepen our interaction with Dr Doitsidou (CIP) on computational sequence analysis of whole-genome sequenced strains of C. elegans.

Exploitation:
In this project, we aim to better understand the fundamental mechanisms of how ion channels act in O2 responses. There is an unmet need to provide better treatment for diseases and disorders linked to ion channels, such as epilepsy, schizophrenia or cancer. Mortality related to brain cancers remains very high, unlike for other cancers where 5-year survival rates have strongly improved. Our fundamental research may inform therapeutic approaches dealing with these channels in brain disorders and cancer in the longer term. The pharmaceutical industry can benefit from our findings by considering new therapies based on our results, and designing new drugs that can target the channels.

Socio-economic benefits:
The high-impact papers resulting from this research will raise the academic and scientific profile of the University and UK research. We use C. elegans nematode worms for this research; doing so offers significant economic and social benefits by replacing vertebrate animal use with a comparatively low-cost invertebrate model.

Communications and Engagement:
We will ensure optimal dissemination of our results to an expert audience by presentations at international and national meetings. I will organise a regional meeting for C. elegans researchers to better connect this growing community.
To ensure efficient communication of our findings to a wider audience, we collaborate with the University press office and Dr Jane Haley, the Edinburgh Neuroscience co-ordinator. C. elegans behaviour is a particularly attractive topic to engage the general public in science and improve their understanding of research. We will engage the public directly within different avenues: My lab has recently started the Model Organism Network (ModON) at the University to strengthen awareness of all organismal resources available at the university and link the researchers using these models. Within this initiative we will organise school workshops in collaboration with Edinburgh Neuroscience. We will also participate in the Edinburgh Science Festival and produce videos about our research.