title: Amiloride-sensitive channels are a major contributor to mechanotransduction in mammalian muscle spindles.
abstract: We investigated whether channels of the epithelial sodium/amiloride-sensitive degenerin (ENaC/DEG) family are a major contributor to mechanosensory transduction in primary mechanosensory afferents, using adult rat muscle spindles as a model system. Stretch-evoked afferent discharge was reduced in a dose-dependent manner by amiloride and three analogues - benzamil, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and hexamethyleneamiloride (HMA), reaching > or = 85% inhibition at 1 mm. Moreover, firing was slightly but significantly increased by ENaC delta subunit agonists (icilin and capsazepine). HMA's profile of effects was distinct from that of the other drugs. Amiloride, benzamil and EIPA significantly decreased firing (P < 0.01 each) at 1 microm, while 10 microm HMA was required for highly significant inhibition (P < 0.0001). Conversely, amiloride, benzamil and EIPA rarely blocked firing entirely at 1 mm, whereas 1 mm HMA blocked 12 of 16 preparations. This pharmacology suggests low-affinity ENaCs are the important spindle mechanotransducer. In agreement with this, immunoreactivity to ENaC alpha, beta and gamma subunits was detected both by Western blot and immunocytochemistry. Immunofluorescence intensity ratios for ENaC alpha, beta or gamma relative to the vesicle marker synaptophysin in the same spindle all significantly exceeded controls (P < 0.001). Ratios for the related brain sodium channel ASIC2 (BNaC1alpha) were also highly significantly greater (P < 0.005). Analysis of confocal images showed strong colocalisation within the terminal of ENaC/ASIC2 subunits and synaptophysin. This study implicates ENaC and ASIC2 in mammalian mechanotransduction. Moreover, within the terminals they colocalise with synaptophysin, a marker for the synaptic-like vesicles which regulate afferent excitability in these mechanosensitive endings.
authors.affiliation: School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK. g.s.bewick@abdn.ac.uk.
authors.forename: Anna
authors.initials: A
authors.lastname: Simon
authors.forename: Fiona
authors.initials: F
authors.lastname: Shenton
authors.forename: Irene
authors.initials: I
authors.lastname: Hunter
authors.forename: Robert W
authors.initials: RW
authors.lastname: Banks
authors.forename: Guy S
authors.initials: GS
authors.lastname: Bewick
mesh_terms: D000200:Action Potentials / Q000502:physiology*; D000344:Afferent Pathways / Q000502:physiology; D000818:Animals; D002478:Cells, Cultured; D053503:Epithelial Sodium Channels / Q000502:physiology*; D015640:Ion Channel Gating / Q000502:physiology*; D008297:Male; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D009470:Muscle Spindles / Q000502:physiology*; D018482:Muscle, Skeletal / Q000294:innervation* / Q000502:physiology*; D051381:Rats; D012026:Reflex, Stretch / Q000502:physiology
chemical_list: D053503:Epithelial Sodium Channels