Introduction UNC-105, and UNC-8 [6,12,18,28,44]. These proteins are
Brain Research 884 2000 1–12 www.elsevier.com locate bres
Research report
Localization of b and g subunits of ENaC in sensory nerve endings in the rat foot pad
H.A. Drummond, F.M. Abboud, M.J. Welsh
Howard Hughes Medical Institute , University of Iowa College of Medicine, 500 EMRB, Iowa City, IA 52242, USA
Accepted 8 August 2000
Abstract
The molecular mechanisms underlying mechanoelectrical transduction and the receptors that detect light touch remain uncertain. Studies in Caenorhabditis elegans suggest that members of the DEG ENaC cation channel family may be mechanoreceptors. Therefore,
1
we tested the hypothesis that subunits of the mammalian epithelial Na channel ENaC family are expressed in touch receptors in rat
hairless skin. We detected bENaC and gENaC, but not aENaC transcripts in cervical and lumbar dorsal root ganglia DRG. Using immunofluorescence, we found bENaC and gENaC expressed in medium to large lumbar DRG neurons. Moreover, we detected these two
subunits in Merkel cell–neurite complexes, Meissner-like corpuscles, and small lamellated corpuscles, specialized mechanosensory structures of the skin. Within these structures, bENaC and gENaC were localized in the nerve fibers believed to contain the sensors
responsive to mechanical stress. Thus b and gENaC subunits are good candidates as components of the molecular sensor that detects touch.
2000 Elsevier Science B.V. All rights reserved.
Theme : Sensory systems
Topic : Somatic and visceral afferents
Keywords : Mechanoreceptor; Mechanosensation; DEG ENaC; Meissner corpuscle; Merkel cell–neurite complex; Pacinian corpuscle
1. Introduction UNC-105, and UNC-8 [6,12,18,28,44]. These proteins are
part of the DEG ENaC protein family that includes the
1
The sensation of light touch is an essential part of the epithelial Na
channel ENaC [4,5,30]. The family also physical perception of the environment. It is required for
includes other proteins that serve as sensors, including the detection of objects and discrimination of shape and
acid-sensing ion channels and the FMRFamide-activated
1
texture. Many studies have described the mechanosensitive Na
channel, FaNaCh [7,16,26,39,46]. DEG ENaC pro- structures that are responsible for light touch sensation; in
teins form non-voltage gated cation channels that in the skin these include Merkel cells, Meissner corpuscles,
several cases
are inhibited
by amiloride
Pacinian corpuscles, Ruffini receptors, and free nerve [4,5,8,15,27,30,43]. These channels are composed of
endings [3,10,29,35]. The stimuli that activate these struc- multiple subunits, as homo- or hetero-multimers. They
tures and their neuronal output have also been reported. each have intracellular amino- and carboxy-termini, two
However, the molecular mechanisms for mechanoelectrical membrane spanning domains, and a large, cysteine-rich,
signal transduction and the receptors that detect mechani- extracellular domain that is thought to serve as a sensor or
cal stress have not been identified. receptor for extracellular stimuli.
Genetic studies in Caenorhabditis elegans have iden- Evidence that DEG ENaC cation channels play an
tified genes for several proteins that may function as important role in mechanosensation first came from the
mechanosensors; these include DEG-1, MEC-4, MEC-10, finding that mutation of several C
. elegans family mem- bers disrupts touch sensation and or coordinated activity in
the worm [6,12,18,28,44]. This result suggested DEG
Corresponding author. Tel.: 11-319-335-7619; fax: 11-319-335-
ENaC channels may serve as mechanosensors. The finding
7623. E-mail address
: mjwelshblue.weeg.uiowa.edu M.J. Welsh.
that another DEG ENaC channel, Pickpocket, was re-
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 8 3 1 - 6
2 H
stricted to multiple dendritic mechanosensory neurons in named hgN-1, a glutathione S-transferase GST fusion
the Drosophila embryo also suggested a central role in protein containing the last 34 amino acids of the
mechanosensation [1,9]. We recently showed that the g C-terminus
of rgENaC
residues 616–649,
subunit of ENaC was located in baroreceptor nerve termi- GSTVPGTPPPRYNTLRLDRAFSSQLTDTQLTNEL na-
nals that innervate the aortic arch and carotid sinus [13]. med rgC-2, and a GST fusion protein containing the
Moreover, amiloride and its analog benzamil attenuated N-terminus of hbENaC HVKKYLLKGLHRLQKGPGY-
baroreceptor function. These data suggest that the g TYKELLVWYCDNTNTGHPKRIICEGPKK
named subunit of ENaC, and possibly other subunits, may form
hbN-1. The antibodies were generated in sheep by Elmira part of the mechanosensory complex. This led us to
Biologicals Iowa City, IA. Immunoreactive sera was consider the possibility that ENaC subunits may also
purified using cyanogen bromide-activated sepharose 4B contribute to peripheral mechanosensation in the skin. To
Pharmacia, Piscataway, NJ linked to the appropriate begin to evaluate this hypothesis, we asked if ENaC
antigen gN-terminus peptide, GST-bN-terminus or GST- subunits are located in touch receptors in the hairless skin
gC-terminus. Detection of ENaC subunits and antibody of the rat paw.
specificity were evaluated in COS-7 cells transfected with both human and rat a, b, and g ENaC [30]. Note that the
N-terminus of rat and human gENaC are 90 identical, the C-terminus of rat and human gENaC are 76 identical,