(Received for publication, February 5, 1996; and in revised form, March 10, 1996)
From the
Mutations of the degenerins (deg-1, mec-4, mec-10) are the major
known causes of hereditary neurodegeneration in the nematode Caenorhabditis elegans. We cloned a neuronal degenerin (MDEG)
from human and rat brain. MDEG is an amiloride-sensitive cation channel
permeable for Na, K
, and
Li
. This channel is activated by the same mutations
which cause neurodegeneration in C. elegans. Like the
hyperactive C. elegans degenerin mutants, constitutively
active mutants of MDEG cause cell death, suggesting that gain of
function of this novel neuronal ion channel might be involved in human
forms of neurodegeneration.
Death of specific neurons is characteristic of many human forms
of neurodegeneration such as Alzheimer's disease,
Huntington's disease, amyotrophic lateral sclerosis, cerebellar
ataxias, and parkinsonism (for review, see (1, 2, 3, 4) ). While some of the
defective genes are known, many have yet to be identified. The
primitive neuronal network of the nematode Caenorhabditis elegans has proven to be a good model of neuronal development and neuronal
death. Hereditary neurodegeneration in C. elegans can be
caused by mutations of the degenerins deg-1(5) ,
mec-4(6, 7) , and mec-10(8) . Homologies with
the amiloride-sensitive Na channel
subunits(9, 10, 11, 12, 13, 14, 15, 16) ,
the functional expression of epithelial Na
channel/mec-4 chimeras(17) , and the vacuolic swelling of
dying neurons(5, 6, 7) suggest that the
degenerins are ion channels and that gain of function is the cause of
neurodegeneration. We report the cloning of a neuronal degenerin from
human and rat brain, a novel ion channel that is activated by mutations
which cause neurodegeneration with the C. elegans degenerins.
Figure 2: Tissue distribution and ontogenesis of MDEG mRNA. A, expression of MDEG mRNA in human tissues. B, expression of MDEG mRNA in rat brain, hippocampal astrocytes, and glial cells from whole brain. C, ontogenesis of MDEG expression in rat brain.
To identify possible mammalian degenerins, we compared the
sequences of deg-1, mec-4, and mec-10 with the EST (expressed sequence
tags) data base and found one matching sequence from brain. We used
this partial sequence to clone the mammalian degenerin homologue (MDEG)
from human and rat brain. The cDNAs from both species code for proteins
of 512 amino acids (Fig. 1) that have all the hallmarks of the
amiloride-sensitive Na channel/degenerin family. Two
hydrophobic regions flank cysteine-rich domains that were shown to be
extracellular for the epithelial Na
channel (24) . The homology with the other members of this ion channel
family is rather low (20-29% identity). Despite the evolutionary
distance between the species, phylogenetic analysis places MDEG closer
to the degenerins of C. elegans and to a recently cloned
molluscan amiloride-sensitive FRMF-amide-gated neuronal Na
channel (25) , than to known mammalian Na
channel subunits (Fig. 1C).
Figure 1:
Deduced protein sequence of MDEG and
comparison with other members of this ion channel family. A,
alignment of human MDEG with the FRMF-amide-gated Na channel from Helix (FaNaCh) and the C.
elegans degenerins. For mec-4 and mec-10, only the sequence of the
second hydrophobic region is shown. Residues identical with or similar
to the corresponding amino acid in MDEG are printed white-on-black or black-on-gray background, respectively. The
hydrophobic regions (MI, MII) of MDEG are labeled
with boxes. The part of MII, thought to line the ionic pore of
the amiloride-sensitive Na
channel (17) and
the degenerins(7) , is hatched. The amino acid that, after
mutation, causes neurodegeneration with the C. elegans degenerins (5, 6, 7, 8) is
marked with a skull and crossbones. Rat MDEG differs from the
human protein in the following amino acids: Ser
Thr, Ile
Leu, Asp
Glu,
Val
Met, Thr
Ala. B,
schematic presentation of structural and sequence homologies. Black
shading indicates similar amino acids. C, phylogenetic
tree.
The MDEG mRNAs of 4.2 and 2.9 kilobases are abundant in brain but were not detectable in any of the other tissues examined (Fig. 2A). MDEG appears to be specific for neurons. It is well expressed in hippocampal neurons and absent in glial cells (Fig. 2B). The mRNA appears just before birth, reaches maximal levels after birth, then declines slightly until adulthood (Fig. 2C).
MDEG
did not induce detectable channel activity after expression in Xenopus oocytes or HEK293 cells. However, MDEG is activated by
the same mutations that cause gain of function in the C. elegans degenerins and
neurodegeneration(5, 6, 7, 8) .
Replacement of Gly (marked with a skull and
crossbones in Fig. 1A) by amino acids bulkier than
Ser activated the MDEG channel. Remarkably, the cutoff for activation
was identical with that reported for mec-4 (6) (Table 1).
All gain of function mutants discriminated poorly between
Na
, K
, and Li
(P
/P
= 2.8 to 5.6,
P
Figure 3:
Properties of MDEG gain of function
mutants. A-C and E-G, MDEG G430V
expressed in Xenopus oocytes. D, MDEG G430F expressed
in HEK293 cells. A, effect of amiloride (100 µM)
on the current recorded from an outside-out patch at -100 mV. B, dose-response curves for amiloride, benzamil, and
ethyl-isopropyl-amiloride on the whole cell current at -70 mV. Points and error bars represent means ± S.E.
for 3 to 5 oocytes. C and D, amiloride (100
µM)-sensitive currents induced by voltage ramps from
-100 to +80 mV from an outside-out patch excised from an
oocyte with Na or Li
in the external
medium (C) or from a whole HEK293 cell with Na
in the external medium (D). E, single-channel
recordings from a cell-attached patch recorded at different potentials
with 140 mM Na
in the pipette solution. F, mean i-V relationships determined on
cell-attached patches with 140 mM Na
or
Li
in the pipette. G, voltage dependence of
the open probability determined on cell-attached patches with 140
mM Na
in the pipette solution. Points and error bars represent means ± S.E. from three
different oocytes.
It seems unlikely that amino acid 430
lines the ionic pore, because the channel pore properties (selectivity,
conductivity) were not altered much by the introduction of a positive
charge (Lys) in this position (Table 2). The activation of MDEG
by bulky amino acids is probably due to steric hindrance. In the model
presented in Fig. 4B, the MDEG sequence flanking
Gly would be part of an inhibitory domain and channel
opening would be caused either by steric constraints (for the gain of
function mutants) or by activation by as yet unidentified mechanisms
(for the wild type channel).
Figure 4: Gain of function mutants of MDEG kill cells. A, HEK293 cells transfected with either wild type or MDEG G430F 20 h after transfection. B, model for the wild type channel blocked by an inhibitory domain and gain of function.
The MDEG channel is inhibited by
mutations that inactivate the C. elegans degenerins deg-1 (26) and mec-4(7) . Replacement of the conserved
Ser by Phe in MDEG G430F results in a completely inactive
channel. No amiloride-sensitive current could be detected in oocytes
injected with 5 ng of MDEG G430F/S443F cRNA (n = 4, not
shown).
Constitutively active MDEG kills oocytes and mammalian cells. Xenopus oocytes injected with either gain-of-function MDEG mutant start to maturate and die (not shown). HEK293 cells transfected with MDEG G430F swell and die (Fig. 4), a mode of cell death also reported for the degenerin-induced neurodegeneration in C. elegans(5, 6) .
Human and rat MDEG
differ only in five amino acids, suggesting a high evolutionary
pressure and an important role in neuronal function. The phylogenetic
neighbors and the structure of MDEG (Fig. 1) provide some
indications about the possible physiological role of this ion channel.
The degenerins mec-4 and mec-10 are required for mechanotransduction (6, 8) , and it has been suggested that they could be
part of a mechanosensitive
channel(7, 8, 9, 26, 27) .
In contrast, the degenerin deg-1 is not involved in
mechanotransduction(5) . MDEG is expressed in hippocampal
neurons where no Na-permeable mechanosensitive ion
channel has been reported yet. We also failed to detect any activation
of MDEG by stretch. We favor the hypothesis that MDEG is a ligand-gated
channel because: (i) the closest homologue of MDEG is the
FMRF-amide-gated channel from Helix(25) , (ii) most of
the MDEG channel protein is located extracellularly in the currently
accepted structural model for this type of proteins(24) ,
suggesting regulation by extracellular signals, (iii) a similar
topology has also been proposed for another ligand-gated ion channel,
the ionotropic purinergic receptor P2x(28) .
FMRF-amide (29) (30 µM), the two known mammalian FMRF-amide like peptides (30) (F8Fa, F18Fa, 30 µM), and the neurotransmitters ATP, glutamate, and acetylcholine (all at 100 µM) failed to activate MDEG, but other neuropeptides or neurotransmitters might be the physiological activators of this novel neuronal ion channel.
So far, C. elegans has proved a valuable animal model for studying neuronal development and neuronal death. Pathways controlling programmed cell death in C. elegans have their counterpart in vertebrates (e.g. the ced-3/ICE (31) and the ced-9/bcl2 (32) connection). On one hand, gain of function of the putative degenerin channels causes degenerative death of neurons in C. elegans, and, on the other hand, excessive activation of cation channels (e.g. the glutamate-gated channels) is involved in human neurodegeneration(33) . Preventing MDEG in mammals and the degenerins in C. elegans from being constitutively activated is a life and death matter for neurons, and gain of function of MDEG may be involved in human forms of neurodegeneration just as constitutive degenerin activity is involved in neurodegeneration in C. elegans. This could be caused either by mutation of the channel, as shown here and for the C. elegans degenerins (5, 6, 7, 8, 26) , or by excessive activation.