©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
The Mammalian Degenerin MDEG, an Amiloride-sensitive Cation Channel Activated by Mutations Causing Neurodegeneration in Caenorhabditis elegans(*)

(Received for publication, February 5, 1996; and in revised form, March 10, 1996)

Rainer Waldmann Guy Champigny Nicolas Voilley Inger Lauritzen Michel Lazdunski (§)

From the Institut de Pharmacologie Moléculaire et Cellulaire, UPR411 CNRS, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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.


MATERIALS AND METHODS

Cloning of the cDNAs

A fragment of the expressed sequence tag (GenBank accession Z45660) was amplified by PCR (^1)and used to screen a rat brain cDNA library (Stratagene). A clone of 2.6 kilobases was sequenced on both strands. The human cDNA was obtained by PCR with Pwo Polymerase (Boehringer) on human brain cDNA using a primer positioned at base 56 of the rat clone (CAGGCTCTCAGGATAACT) and a degenerate primer flanking the stop codon (TCARCANGCDATYTCYTCNAG) and sequenced on both strands. For both species, an open reading frame of 1536 bases was preceded by stop codons in all three frames.

Primary Cultures of Neurons and Glial Cells

Primary cultures of rat hippocampal neurons (embryonic day 17-18, 5 days in culture) and of hippocampal astrocytes (3-4-day-old rats, 3 weeks in culture) were prepared as described(18) . Glial cells from adult rat brains were prepared and cultured as described(19) .

RNA Isolation and Northern Blots

Human multitissue Northern blots containing about 2 µg of poly(A) RNA per lane (normalized for identical beta-actin expression) were purchased from Clontech. For the blots with RNAs from rat, total RNA was isolated as described(20) , 10 µg (Fig. 2C) or 20 µg (Fig. 2B) of RNA in each lane were separated on 1% agarose/formaldehyde gels and transferred onto nylon membranes. The probes were random prime P-labeled and corresponded to bases 1 to 1308 for the human probe and to bases 217 to 1363 for the rat probe (positions refer to the nucleic acid sequences submitted to GenBank). The blots were hybridized overnight at 65 °C in 5 times SSC, 10 times Denhardt's solution, 0.1% SDS, 100 µg/ml herring sperm DNA, washed with 0.1 times SSC, 0.1% SDS at 70 °C and subsequently exposed to Kodak X-Omat AR film for 3 to 5 days at -70 °C. The sizes of the mRNAs were calculated relative to the markers on the commercial blot and relative to the mobility of the ribosomal RNAs for the rat mRNAs. For the blots with rat RNA, hybridization with a glyceraldehyde-3-phosphate dehydrogenase probe gave similar signals for all lanes of each panel in Fig. 2.


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.



Construction of Expression Vectors and Mutagenesis

Noncoding sequences were removed by PCR amplification with the primers AGAATTCGCCGCCACCATG and ATCTCGAGTCAGCAGGCAATCTCCT, and the EcoRI/XhoI-digested PCR product was subcloned into the pBSK-SP6-Globin vector(15) . Mutants of the rat clone were prepared as described (21) and sequenced. For expression in mammalian cells, the cDNAs were excised from the pBSK-SP6-Globin vector with EcoRI/XhoI and subcloned into the EcoRI/SalI-digested PCI expression vector (Promega).

Expression and Electrophysiological Analysis

For expression in Xenopus oocytes, cRNA was synthesized from the NotI digested vector using a kit from Stratagene. Xenopus oocytes were injected with 5 pg of cRNA and used 1-3 days after injection. For mutants that were inactive at this concentration, 5 ng of cRNA were injected to confirm their inactivity. HEK293 cells were transfected with the MDEG-PCI constructs using LipofectAMINE (Life Technologies, Inc.) following the manufacturer's protocol and used for electrophysiology after 8-20 h. Xenopus oocytes and HEK293 cells were maintained in the presence of 100 µM amiloride prior to electrophysiological analysis. Oocyte injection, microelectrode voltage clamp, and patch clamp recordings were essentially carried out as described(22, 23) . The bath solution for outside-out patches and the pipettes for cell-attached patches contained 140 mM NaCl (or LiCl), 1 mM MgCl(2), 1 mM CaCl(2), 10 mM Hepes (pH 7.4). For outside-out patch and whole-cell recordings, pipettes contained 140 mM KCl, 2 mM MgCl(2), 5 mM EGTA, 10 mM Hepes (pH 7.4). Data were filtered at 100 Hz and analyzed using Biopatch software (Biologic). All expression studies were performed with the clone from rat.

Computer Analysis

The MACAW program (NCBI) was used for the multiple sequence alignments and the schematic presentation of homologies. The phylogenetic tree was calculated with the GCG software (Genetic Computer Group, University of Wisconsin) using Kimura correction for multiple substitutions and the UPGMA option. The Blast network server (NCBI) was used for all data base searches.


RESULTS AND DISCUSSION

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.


FOOTNOTES

*
This work was supported by CNRS and the Association Française contre les Myopathies (AFM). Bristol-Myers Squibb Co. supplied an ``Unrestricted Award.'' The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 33-93-95-77- 00 or 02; Fax: 33-93-95-77-04. douy{at}.unice.fr.

(^1)
The abbreviations used are: PCR, polymerase chain reaction; alphaNaCh, betaNaCh, NaCh, NaCh, amiloride-sensitive Na channel alpha, beta, , and subunits, respectively.


ACKNOWLEDGEMENTS

We thank Drs. Jacques Barhanin and E. Lingueglia for helpful discussions, Martine Jodar and Nathalie Leroudier for skillful technical assistance, and Frank Aguillia for help with the artwork.


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©1996 by The American Society for Biochemistry and Molecular Biology, Inc.