©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Degenerin-containing Chimeras Identify Residues Essential for Amiloride-sensitive Na Channel Function (*)

Rainer Waldmann , Guy Champigny , Michel Lazdunski (§)

From the (1) Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The highly selective, amiloride-sensitive Na channel is formed of three homologous subunits termed , , and . The three subunits exhibit similarities with Caenorhabditis elegans proteins called degenerins involved in sensory touch transduction and, when mutated, in neurodegeneration. Swelling of neurons observed in neurodegeneration suggests an involvement of ion transport, but the channel function of degenerins has not yet been demonstrated. We used chimeras to study the functional relationship between the epithelial sodium channel and the degenerin Mec-4. Exchange of the hydrophobic domains of the Na channel subunit by those of Mec-4 results in a functional ion channel with changed pharmacology for amiloride and benzamil and changed selectivity, conductance, gating, and voltage dependence. All of these differences were also obtained by exchanging Ser-589 and Ser-593 in the second transmembrane region by the corresponding residues of Mec-4, suggesting that these two residues are essential for the ionic pore function of the channel.


INTRODUCTION

Sodium transport through the epithelial Na channel (NaCh)() is the limiting step for Na reabsorption in various tight epithelia (1, 2) . NaCh is also involved in taste perception (3) .

Expression cloning of the epithelial sodium channel demonstrated that it was made of at least three homologous subunits called , , and (4, 5, 6, 7, 8) . While these three subunits show no significant sequence homology with other previously cloned channels, significant similarities have been found with members of the Caenorhabditis elegans degenerin family (such as Mec-4, Mec-10, and Deg-1) (9-12), a set of proteins associated with the mechanosensory apparatus that can be mutated to cause neuronal degeneration. Degenerins may function as channel proteins, but this has not been demonstrated yet.

The NaCh subunits and the degenerins have two hydrophobic regions long enough to span the membrane, and both might participate in the formation of the ionic pore (8, 13, 14) . To evaluate their specific roles, chimeras between the subunit of the Na channel (NaCh) and Mec-4 were constructed and studied after co-expression with the and subunits in oocytes. NaCh and Mec-4 were selected because NaCh plays a key role in the formation of NaCh (4, 5, 6, 7) and Mec-4 in mechanotransduction and neurodegeneration (10).


EXPERIMENTAL PROCEDURES

Goldman, Engelman, and Steitz scores for hydrophobic transbilayer helices were plotted using the GCG sequence analysis software, and the putative transmembrane regions were selected.

Mutants were prepared by site-directed mutagenesis using synthetic oligonucleotides (15) and verified by dideoxy sequencing.

cRNAs were prepared and oocytes were injected with 100-500 pg of either NaCh or mutant NaCh cRNA together with the same amount of cRNA for and NaCh as described (16) .

Cell-attached recordings were performed on oocytes clamped to zero mV in high K medium. Pipettes contained (in mM): NaCl (or LiCl) 140, MgCl 1, CaCl 1, Hepes 10, pH 7.4. Data were sampled at 1 kHz and filtered at 300 Hz for analysis. Single-channel conductances for Na and Li were calculated from I-V relationships from 0 mV to -100 mV with Na or Li as conducting ion. Open probabilities and mean dwell times were calculated with Biopatch software (Biologic). Values are means ± S.E.


RESULTS AND DISCUSSION

The first (MI) or the second (MII) hydrophobic region of NaCh were replaced by corresponding sequences of Mec-4 (Fig. 1a). Exchange of either hydrophobic domain caused significant changes in conductance, gating, and pharmacology. Affinity for the diuretic amiloride (Fig. 1c) and for benzamil (), a specific NaCh blocker, decreased by a factor of 3 after exchange of MI and by a factor of 14 after replacement of MII.


Figure 1: NaCh/Mec-4 chimeras. a, alignment of the putative transmembrane domains MI and MII of NaCh and Mec-4. Boxes indicate sequences of NaCh that were replaced by the corresponding Mec-4 residues. Ala-442 in MII of Mec-4, which causes neurodegeneration after mutation, is underlined. The symbols used in b-d are: , NaCh; , MI chimera; , MII chimera; , MIMII chimera. b, cell-attached recordings at various membrane potentials with Na in the pipette. c, inhibition of the macroscopic current at -70 mV by amiloride. Each point represents the mean value obtained from 5-7 oocytes. d, voltage dependence of open probability. Points represent mean values from 3 (NaCh), 5 (MI chimera), 4 (MII chimera), or 2 recordings (MIMII chimera). e, mean single-channel conductance for Li and Na. ★, significantly different from NaCh, with p < 0.001 using an unpaired t test. The sequences shown for MI of NaCh correspond to amino acids 111-132 (5). MI of Mec-4 is not in the data bases and was obtained by translating the Mec-4 genomic sequence (10) (bases 1623-1688). For MII, the actual hydrophobic region is longer than the approximately 20 amino acids required to span the membrane. Sequences shown are amino acids 567-602 for NaCh (5) and 433-476 for Mec-4 (10).



Replacement of MI caused only a slight although significant increase in single-channel conductance for Na and Li (Fig. 1e). Exchange of MII had more drastic effects on the ionic pore; the conductivity for Na nearly tripled, while that for Li increased only slightly, resulting in reversed selectivity for Na over Li (P


Figure 2: Mutations in MII of NaCh. a, partial alignment of MII of NaCh and Mec-4. Arrows toward the Mec-4 sequence indicate replacement of a residue in NaCh by the corresponding amino acid of Mec-4. Upwardarrows indicate other point mutants of NaCh, and bars mark the sequences of NaCh replaced by corresponding parts of Mec-4. Mutants labeled with had properties indistinguishable from NaCh. The symbols used for b-e are: , S589I; , S593T; , S593I. b, cell-attached recordings at various membrane potentials. c, inhibition of the macroscopic current at -70 mV by amiloride. Each point represents the mean value obtained from 5-7 oocytes. Dashedlines and icons indicate the log of the inhibition constant (pK) values for NaCh and the MII chimera (Fig. 1b), respectively. d, voltage dependence of open probability. Points represent mean values from 9 (S589I), 4 (S593T), or 5 (S593I) recordings. e, single-channel conductance for Li and Na. For ease of comparison, the values for the NaCh and the MII chimera (Fig. 1e) are shown again. f, mean single-channel current-voltage relationships for S593I with different Ca concentration in the Na containing pipette solution (, , ) and for NaCh ().



We tested two chimeras within this part of MII (Fig. 2a, ). Chimera A (Ser-589 to Val-595) had properties identical to those of the MII chimera, while chimera B (Met-597 to Phe-602), despite important sequence changes, had characteristics identical to those of NaCh ().

The amino acids that differ between chimera A and NaCh were replaced one by one in NaCh by their Mec-4 counterparts (Fig. 2a, ). Only two of these mutants (S589I and S593T) had properties different from those of NaCh.

Replacement of Ser-589 by Ile (S589I) caused changes in channel properties nearly identical to those observed with the MII chimera. The S589I mutant had a decreased affinity for amiloride (Fig. 2c, ), a higher conductance for Na than for Li (Fig. 2e, ), a low open probability, and a fast voltage-dependent gating (Fig. 2, b and d, ). The only minor but significant difference was a lower single-channel conductance for both Na (10 pS) and Li (6.4 pS) than the MII chimera (Na: 12.7 pS; Li: 8.3 pS). Mutation of Ser-593 to Thr (S593T) significantly increased the conductance for Na by 2.5 pS and that for Li by 1.3 pS compared to NaCh, leaving the other properties unchanged (Fig. 2, ). Together, these two mutations (S589I and S593T) account for all of the differences in channel properties observed between NaCh and the MII chimera.

Substitution of Ser-589 by Phe, the corresponding residue of the degenerin Deg-1 (11) , caused the same changes in channel properties as did S589I, except for a 3-fold lower Kfor amiloride (8.9 µM) than S589I ().

The mutation S589I had more drastic effects than S593T, but replacement of a Ser by an Ile causes more structural changes than replacement by a Thr. Therefore, we also replaced Ser-593 in NaCh by Ile (S593I). The differences with NaCh increased and included a fast voltage-dependent gating (Fig. 2, b and d, ) and a voltage-dependent block by external Ca (Fig. 2f), suggesting localization of Ser-593 within the ion-conducting pathway, but the affinity for amiloride remained unchanged (Fig. 2c, ). Voltage-dependent block by external Ca was never observed for NaCh (Fig. 2f) and was a typical behavior of the S593I mutant.

Our data suggest that Ser-589 and Ser-593 face the same side of an -helix and line the ionic pore (Fig. 3). Ser-589 is probably located close to the outer mouth of the pore and is important for the fixation of amiloride, which blocks from the outside (1, 2) . Ser-593 is located on the inner mouth of the pore and does not participate in the binding of diuretics.


Figure 3: Topology of MII. The sequence of the amphiphilic part of NaCh MII is shown in uppercaseletters and the corresponding residues of Mec-4 as footnotes within the helix. Residues that differ between NaCh and Mec-4 and cause significant changes in channel properties after mutation are numbered. Arrows indicate the characteristics of the channel that changed after mutation.



Both MI and MII play a key role in the formation of the ionic pore of NaCh and, very likely, that of the degenerins. Our results indicate that Mec-4 is a subunit of a possibly Na-permeable ion channel sharing a common pore topology with NaCh but having very different pharmacological and biophysical properties.

Mutation of Ala-442 in Mec-4 to Thr (Fig. 1a) causes neurodegeneration in C. elegans, and it has been speculated that this could be due to increased ion flow through the putative channel (10) . When introduced into the MII chimera, this mutation (MII chimera A577T; not shown) did not change the properties of the channel. This finding, together with the fact that degeneration can occur long after differentiation of neurons (10) , suggests that this form of neurodegeneration is more likely caused by impaired regulation rather than by an alteration of the basic biophysical properties of the channel.

Other tissues (1, 2, 17) (i.e. endothelial cells (18) and smooth muscle (19) ) contain a family of amiloride-sensitive channels with different Naversus K selectivity, conductance, gating, and pharmacological properties, but none of these channels has been cloned yet. Since small changes in the sequence of NaCh suffice to completely alter channel properties, it seems possible, if not likely, that some of this diversity is caused by alternate splicing or editing of the NaCh mRNAs, as is the case for another multi-subunit ion channel, the ionotropic glutamate receptor (20, 21).

  
Table: Channel characteristics of NaCh and different mutants

The table shows mean values ± S.E. n, number of experiments. Dose-response curves used for Kdetermination were constructed from 5-7 different experiments. P was measured at -60 mV.



FOOTNOTES

*
This work was supported by the CNRS. 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; Fax: 33-93-95-77-04; E-mail: ipmc@unice.fr.

The abbreviations used are: NaCh, epithelial Na channel; NaCh, epithelial Na channel subunit; MI, first transmembrane region of the Na channel or Mec-4; MII, second transmembrane region of the Na channel or Mec-4; P


ACKNOWLEDGEMENTS

We are very grateful to Drs. E. Lingueglia, N. Voilley, and P. Barbry. We thank C. Roulinat and F. Aguila for technical assistance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.