Evaluating DEG/ENaC Channels as the Elusive Mammalian Mechanotransducer

Supervisors: Dr Guy Bewick, Dr Ekkehard Ullner and Dr Zhuoyi Song (UCL)

Background:

Sensing what the body is doing and touching is fundamental to animal behaviour. Yet, mechanosensation is perhaps the least understood of the mammalian senses. Indeed, it is the only one where the detector (transducer) proteins are unidentified. Several suggested candidate channel families (transient receptor potential - TRPs, Piezos, and the degenerin/epithelial sodium channels – DEG/ENaCs) all remain controversial. We propose much of the uncertainty arises from (i) evaluation in culture with (ii) mixed sensory neurone populations that are (iii) immature and lack differentiated sensory terminals, (iv) that whole genome ENaC deletions are lethal, (v) channels may act as heteromers, plus (vi) the diversity and typically small size of mechanosensory endings.

The proposed project’s novelty and timeliness, therefore, lie in:

(a) using only fully differentiated, mature terminals,

(b) using the mammal’s largest mechanosensory endings, the muscle spindle,

(c) targeting candidate channel gene deletions specifically to mechanosensory neurones and d) specifically studying sodium-selective channels, since sodium is the major component of mammalian muscle spindle transducer currents1. Since triple knock out of the ASIC family of DEG/ENaCs has a very mild phenotype, this reasoning spotlights ENaC channels, with the non-selective cation or calcium-selective TRP and Piezo channels playing at most a subsidiary role. We have shown spindles express ENaC2 and TRPV3 and –C1 (pilot data). This proposal will comprehensively screen spindle homogenates for all the DEG/ENaCs, TRPs and Piezos expressed and evaluate, on a triple ASIC knock out mouse background, the functional effects of deleting single additional genes in mechanosensory neurones. A final project aspect will adapt a powerful new ‘white box’ mathematical approach used on the fly visual transducer potential to the mechanosensory potential. Such a general model will be a powerful tool for testing which, if any, of these components are necessary and sufficient to produce the electrophysiological responses generated by stretch and, if not, predict the properties of the missing components.
Experimental design: The project will be based in Dr Bewick’s laboratory in Aberdeen. Using a highly enriched source recently developed in another project, spindle protein homogenates will be made and screened using mass spectrometry (MS) at Aberdeen’s core proteomics facility for all ASIC and ENaC sodium channels, plus TRPs and Peizos. Hits will be validated by immunolabelling in Western blots (WB) and muscle cryosections. Then, the pharmacology of spindle stretch-evoked electrophysiological responses will be probed using the best available channel-selective ligands for the validated candidates. The student will then train with Dr Song (UCL) in the principles of her novel ‘white box’ mathematical model of the visual receptor potential3, which has a strikingly similar profile to the spindle mechanosensory receptor potential. The model adaptation will seek to validate both the generality of the model and specific roles for the primary transducer channels identified above, plus incorporate already identified3 modulatory channels downstream of transducer activation. Finally, deletions of single validated channel hits will be targeted to mechanosensory neurones on a global triple ASIC KO mouse background (Prof Chih-Cheng Chen, Taiwan Mouse Clinic). Spindle responses from these mice will be analysed for disruption to stretch-evoked firing.

We seek to recruit a mathematics/systems modelling graduate with a thorough grasp of relevant mathematical techniques.