(The FASEB Journal. 2002;16:1623-1629.)
© 2002 FASEB
Family ties of gated pores: evolution of the sensor module
ATTILA KUMÁNOVICS,
GAL LEVIN* and
PAUL BLOUNT*1
Center for Immunology and
* Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
1Correspondence: Department of Physiology, University of Texas Southwestern Medical Center, Harry Hines Blvd., Dallas, TX 75390-9040, USA. E-mail: Paul.Blount{at}UTSouthwestern.edu
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ABSTRACT
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The six-transmembrane channels are thought to be composed of two modules: pore and sensor. Whereas the modular design of the pore has been established, the modularity of the sensor remains hypothetical. As a first step toward establishing the modularity of this region, we searched for genes where the sensor is found independent of the pore and have identified new members of the sensor superfamily. Analysis of these sensors reveals a motif shared among not only these newly discovered members and voltage-gated, transient receptor potential, and polycystin channel sensors, but also MscL, a bacterial mechanosensitive channel. Mutational analyses presented here and in previous studies demonstrate that highly conserved residues within this motif are required for normal channel activity; mutations of residues within this motif in different subfamilies lead to consistent channel phenotypes. Previous studies have demonstrated that peptides containing this motif and the adjacent conserved transmembrane domain elicit channel activities when reconstituted into lipid membranes. These data provide evidence for the modularity of the sensor, imply a model for its evolution, suggest a common origin for mechano- and voltage-sensing, and may offer a glimpse of the properties of the first sensor/channel.—Kumánovics, A., Levin, G., Blount, P. Family ties of gated pores: evolution of the sensor module.
Key Words: MscL • TPTE • transient receptor potential • tyrosine phosphatases • voltage-gated channel
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INTRODUCTION
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VOLTAGE-GATED CHANNELS (CHV) with six-transmembrane domains (TMDs), S1-S6, are members of a large gene superfamily that includes the depolarization-activated cation channels, transient receptor potential (TRP) and TRP-like channels (1
, 2)
, polycystins (PKD1, PKD2, PKDL) (3)
, cyclic nucleotide-gated (CNG) channels, and inositol 1,4,5-triphosphate receptors (IP3R) (4)
. All are organized as four subunits per complex, which may be linked into a single polypeptide as found in voltage-gated sodium and calcium channels.
These channels are thought to contain two modules: sensor (S1-S4) and pore (S5-S6). Whereas the evolution and structure of the pore module has been extensively studied, little is known of the origin of the sensor (4
, 5
, 6)
. Here we identify sensor modules that lack sequences encoding the pore. By bioinformatics, we identified conserved motifs and new members of the superfamily of sensory proteins. We experimentally support the prediction that MscL, a bacterial mechanosensitive channel (7)
, is a distant member of the sensor superfamily. Combining our data with previous findings, we hypothesize that the sensor is modular and that the superfamily encompasses genes and gene families beyond what was previously recognized. In addition, we propose an evolutionary history for the sensor module and a model for the structure of the first sensor.
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THE SENSOR IS FOUND INDEPENDENT OF THE PORE
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If the sensor domain is truly modular, it should be present in some proteins independent of the pore. Yet, in the literature, there are no such examples. Hence, we searched sequence databases for proteins that contained the sensor domain independent of the pore. In the Cluster of Orthologus Groups (COG) database (8)
, COG1226 represents the family of six-transmembrane potassium efflux channels (9)
. By searching COG1226 for proteins with fewer than six TMDs, we discovered two independent pairs of genes that may form functional channels. One pair is L136668 (GenBank# AAK05011) and L136943 (GenBank# AAK05012) from Lactococcus lacti (10)
. L136668 encodes the first three TMDs found in voltage-gated channels; L13694, predicted to be the gene for a potassium channel-like protein, encodes the last two TMDs (Fig. 1
). Intriguingly, both these genes lack the charged S4-like TMD that is thought to serve as the primary voltage sensor. The other gene pair is MTH1779 (GenBank# AAB86245) and MTH1778 (GenBank# AAB86244) from Methanobacterium thermoautotrophicum (11)
. MTH1779 apparently encodes a protein similar to the first five TMDs of voltage-gated channels, whereas MTH1778 encodes a short protein with one S6-like TMD (Fig. 1)
. Although the homologies clearly suggest these genes encode potassium-like channels, neither contains Gly-Tyr/Phe-Gly, the canonical motif of the selectivity filter for this family. There are short (65 and 23 bp long) segments between the pairs in each of the loci, but these are not sufficient to encode the missing S4 or pore loop-forming segments. These gene pairs are apparently the result of a genuine gene fission (12)
and not of a sequencing error or simple insertion or deletion mutations. It is likely that these gene products assemble to generate a functional protein complex. The proximity of the genes within the pairs makes it probable that they are components of a functional operon.
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Figure 1. Multiple alignment of selected prokaryotic voltage-gated potassium channel-like proteins from COG1226. The sequence naming follows the nomenclature used in the COG database. A) The multiple alignment was generated by CLUSTALW1.8 (41)
and edited with SEAVIEW (42)
. The amino acid residues are grouped according to 60% consensus (using CONSENSUS at http://www.bork.embl-heidelberg.de/Alignment/consensus.html) and labeled. Hydrophobic: ‘h’ (F, Y, W, L, I, V, M, C, H, K, R, and A); aliphatic residues: ‘l’ (L, I, and V); aromatic residues: ‘a’ (F, H, W, and Y) highlighted in yellow; positive residues: ‘+’ (H, K, and R) highlighted in red; negative residues: ‘-’ (D and E) highlighted in blue; polar residues: ‘p’ (C, D, H, E, K, N, Q, R, S, and T); small residues: ‘s’ (S, A, G, T, V, P, N, C, and D); tiny residues: ‘u’ (G, A, and S); alcohol residues: ‘o’ (T and S); turnlike residues: ‘t’ (A, C, D, E, G, H, K, N, Q, R, S, and T) highlighted in green. The positions that are not conserved at the given level are labeled as ‘.’. Identical residues are shown in white letters on black. Approximate locations of the transmembrane segments are labeled above the alignment. B) Schematic representation of the membrane structure of the putative channels from L. lacti and M. thermoautotrophicum. In L. lacti, orf L136668 encodes a protein similar to the first three TMDs of the 6TM-KVs and L136943 encodes the last two TMDs. There is no S4-like TMD in this putative channel. In M. thermoautotrophicum orf MTH1779 encodes a protein similar to the first five transmembrane segments of a 6TM-KV, whereas MTH1778 encodes a short protein with one S6-like TMD.
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Additional PSI-BLAST searches with the membrane portion of the mouse TRP6 (amino acid residues 400–800, GenBank# Q61143) using various matrices and gap penalties repeatedly identified TPTE (transmembrane phosphatase with tensin homology), a recently described novel testis-specific protein family conserved among mammals (13
, 14)
. By comparing the amino-terminal 220 amino acid portion of the TPTE protein to the nonredundant protein data set of GenBank by iterative PSI-BLAST searches, the family of voltage-gated cation (sodium, calcium, and potassium) channels was identified with high a degree of confidence (E<0.001) within the first three iterations. Further iterations recognized all other types of six-transmembrane channels (6TM) such as polycystins, TRP, IP3 receptors, and CNG channels.
Aligning the TPTE protein with the voltage-gated cation channels corroborates all three TMDs predicted (13)
and predicts a fourth (Fig. 2
). The fourth membrane-spanning region contains five positively charged residues and aligns with the fourth, S4, voltage-sensing TMD of the voltage-gated 6TMD channels (Fig. 2)
. Note that the pore region of voltage-gated channels is replaced with a tensin-like tyrosin phosphatase domain in the TPTE protein.
The eukaryotic TPTE represents a perfect sensor module-alone configuration. The function of this unique protein is not known (16)
. Whether the protein forms a pore, interacts with a pore domain, or the phosphatase activity is regulated by voltage remains a viable question.
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THE TIES THAT BIND ...
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As the next step toward addressing the question of functional origins, we sought to resolve the most conserved regions within this module and determine whether more distant members could be found. Clearly, S3 appeared to be the most preserved (Fig. 3
A),with more conserved residues than the P-loop, which is ion-specific, or even the S4 domain. The aspartate of a much-conserved Asn-hydrophobic-hydrophobic-Asp (N-h-h-D) motif is the most invariant amino acid in the entire superfamily (Figs. 1
, 2
, and 3)
. The N-h-h-D motif is shared among essentially every group and every homologue of potassium, calcium and sodium voltage-gated channels, most members of the TRP and polycystin families, and the putative melastatin and MTR1 channels (17
18
19)
. In channels where the sensor module is thought to be vestigial, such as the ligand-gated CNG and IP3R channels, this motif is not conserved (data not shown).
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Figure 3. Conserved sequences from the sensor module A) Multiple alignment of the third segments (S3) of voltage-gated cation channels. The amino acid residues are grouped according to 70% consensus. The alignment and residue coloring is the same as in Fig. 1
. Accession numbers are as follows: TRPs are blue: TRP_DROME: P19334; TRPL_DROME: P48994; TRP1_HUMAN: P48995, TRP2_MOUSE: NP_035774; TRP3_HUMAN: Q13507; TRP4_HUMAN: Q9UBN4; TRP5_HUMAN: Q9UL62; TRP6_HUMAN: Q9Y210; 6TM-CaV are pink: CCAD_HUMAN: Q01668; CCAA_DROME (or CACAPHONY): P91645; 6TM-NaV are green: SCN2A1_HUMAN (or CINA): Q99250; SCNA_DROME (or CINA or PARALYTIC): P35500; 6TM-KV are purple: KCNA1_HUMAN (or CIK1): Q09470; KCNA4_HUMAN (or CIK4): P22459; KCNA5_HUMAN (or CIK5): P22460; KCNC1_HUMAN (or CIKD): P48547; KCNC4_HUMAN (or CIKG): Q03721; KCNB1_HUMAN (or CIKA): Q14721; KCNB2_HUMAN (or CIKB): Q92953; SHAKER_DROME: P08510. B) Multiple alignment of the first transmembrane segment (TM1) of MscL channels (names printed in red) from Gram- and Gram+ bacteria with the third transmembrane segment (S3, names printed in blue) of the Drosophila TRP and human TRP-like channels. The MscL family was identified using HMMER 2.1.1 (43)
; the model was built as a single local (Smith-Waterman) alignment using the following arguments: hmmbuild--s--archpri 0.3--sswentry 0.8--swexit 0.2. The model was calibrated at fixed length (--fixed 19) and for the database search the post hoc second null model was turned off (--null2). Membrane proteins of the SwissProt database (44)
, release 39.20, were used for these searches. The alignment, grouping and residue coloring is the same as above. Labels above the sequences (* +) indicate residues identified as important for normal gating by mutagenesis studies (20)
, as described in text. The sequence naming follows the SWISS-PROT nomenclature: VCHOL, Vibrio cholerae; ECOLI, Escherichia coli; BACSU, Bacillus subtilis; XFAST, Xiella fastidiosa; HUMAN, Homo sapiens; DROME, Drosophila melanogaster.
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Next, we built a similarity matrix based on the most highly conserved S3 segment. An HMMER search identified all of the genes described above and, surprisingly, also the bacterial large-conductance mechanosensitive channel (MscL) family (7)
. The homology to MscL has a high degree of confidence (E<0.001) with the core of the similarity at the cytoplasm–membrane interface of S3 (TMD1 of MscL). Furthermore, MscL contains the N-h-h-D motif (Fig. 3A, B
).
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MUTAGENIC STUDIES SUPPORT THE HYPOTHESIS THAT MSCL BELONGS IN THE SENSOR SUPERFAMILY
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The functional importance of this conserved domain has been demonstrated in a random mutagenesis study of Escherichia coli MscL selecting for gain-of-function (GOF) slowed-growth phenotypes. The mutated channels gated at a lower membrane tension (20)
. Significantly, 14 of the 18 mutations at 9 of the 13 mutated residues were found in, or just cytoplasmic to, M1 (marked with an * or + in Fig. 3B
); many of these residues are either highly conserved (E. coli N15, marked in Fig. 3B
with an *) or limited largely to conserved substitutions (L19, V21, G22, V23, G26, G30; Fig. 3
) within the superfamily. The N15 of MscL is the asparagine in the N-h-h-D motif. Remarkably, an analogous mutation in Kv2.1 from an independent subfamily also led to a GOF channel requiring 22 mV less stimulus to gate half-maximal (21)
.
In several studies the conserved N-h-h-D aspartate (marked in Fig. 3B
by an ), has been mutated and the resulting channel activities studied. The Shaker D316 has been shown to be critical for channel activity (22
, 23)
; further studies suggested that it electrostatically interacts with a positively charged residue within S4 (24)
; it is currently unclear whether this electrostatic interaction occurs in the closed, open, or both states of the channel. The analog D258 of the delayed rectifier Kv1.1 potassium channel abolished ionic current; even a charge conservative glutamate mutation was not tolerated (25
, 26)
. Mutation of the D262 in Kv2.1 led to channels that required an additional 45mV to achieve half-maximal currents (21)
. Finally, mutation in the conserved aspartate in the human PKD2 protein (which is D511) segregated with the autosomal polycystic kidney disease in one family investigated (27)
. Because complete channel loss is required for the polycystic kidney disease pathogenesis, this mutation represents a true missense variant.
If MscL truly is a member of the superfamily, then mutation of the corresponding amino acid in MscL, D18, should also lead to channel dysfunction. We therefore substituted it to cysteine and assayed for channel function. We used a strain deficient in MscL and YggB, two genes contributing to the E. coli major mechanosensitive channel activities, leaving the strain unusually sensitive to sudden decreases in osmotic environment (28
, 29)
. Unlike wild-type E. coli MscL, the D18C mutant did not rescue the cell death phenotype (Fig. 4
). These data demonstrate that the D18C-mutated MscL is not functional in vivo.
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Figure 4. The E. coli MscL containing a D18C mutation does not function in vivo as determined by phenotypic suppression. The mscL and yggB double null strain, MJF455, transformed with either the pB10 expression plasmid (vector) or pB10 containing wild-type E. coli MscL (expressed WT) or the E. coli MscL D18C mutant, was subjected to a 1000-fold down shock in sterile water for 20 min. The parental strain, FRAG-1, which contains both mscL and yggB was also tested. Viability, as derived previously (29)
, is shown with SE from 5 or more independent experiments.
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To determine whether the D18C mutated MscL protein possessed mechanosensitive channel activity, giant spheroplasts were generated and channel activity was assayed in native bacterial membranes by patch-clamp (30)
. Activity was rarely observed and then only close to lytic membrane tensions (not shown). Using MscS, the other major E. coli mechanosensitive channel activity, as an internal control (30)
, we estimate that nearly twice as much tension is required to gate the mutated channel compared to wild-type MscL. No increase in channel activity was observed in the presence of 5 mM DTT, suggesting that lack of activity was not due simply to disulfide bridging. Similarly, addition of the sulfhydryl reagent MTSES, which should restore the negative charge at this site, did not restore activity. These data are not merely due to decreased expression; a previous study demonstrated that this mutant expresses at normal levels (31)
.
The mutational data above support the hypothesis that MscL is a sensor superfamily member. They also demonstrate a correlation between mutational site and channel phenotype: mutation of the asparagine leads to a channel more sensitive to stimuli in two superfamily members from distinct subfamilies, whereas mutation of the aspartate results in a partially or fully nonfunctional channel in five independent superfamily members from three distinct subfamilies.
The crystal structure of an MscL protein may provide insight into channel function (see Fig. 5
). Although D18 of MscL does not appear to interact with a positively charged residue in the closed state, it is possible that such an interaction occurs in the open conformation; alternatively, it may interact with zwitterionic lipids in either or both states. With a great deal of information on structure and molecular mechanisms (7)
, MscL has been and remains a relevant model system for studying sensing.
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Figure 5. Locations of the most conserved residues are shown within a structural model of the M. tuberculosis MscL as derived from X-ray crystallography. The cytoplasmic view (45)
(left) and side view (right) of this homopentameric molecule are shown. The lines in the side-view presentation indicate the approximate location of the membrane. The highly conserved residues of the N-h-h-D motif are shown: purple residues are analogous to N15 and the green residues are analogous to D18 in the E. coli MscL.
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A PROPOSAL FOR THE COMMON EVOLUTION OF SENSING VOLTAGE AND MEMBRANE TENSION
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The common ancestry of mechano- and voltage-gated channels may stem from the relationship between membrane tension, curvature, and electrical potential. Previous studies have shown that an electrical gradient across a membrane changes the membrane curvature and can result in membrane tension in a patch-clamp pipette as well as whole cells (32
, 33)
. Conversely, forcing a membrane curvature induces a voltage potential across a membrane (known as flexoelectricity) (34)
. Therefore, the first sensors may have developed from simple homomultimer single TMDs that sensed effects of electrochemical gradients on the membrane or, indirectly, cell volume by determining the curvature of the membrane. We propose that this simple primordial sensor was similar to the conserved segment of the sensor superfamily (Fig. 6
). Most likely this primordial sensor moved or rotated in response to stimuli and either opened a pore or stimulated other factors or peptides. Since that time, many channels appear to have specialized into either voltage- or mechanosensors. We speculate that whereas MscL kept a streamlined form and developed or maintained the ability to gate a pore in response to membrane tension (28)
and heat (35)
, the sensor module of voltage-gated channels became fine-tuned for voltage sensing and attached to preexisting pores. Many members of the TRP family are similar to MscL in that they sense heat, changes in osmolarity, and even mechanical stimuli (2)
; one member has recently been shown to respond to cold stimuli and menthol (36
, 37)
. If voltage- and mechanosensors have a common origin, examples of channels with both mechano- and voltage-sensitive properties are likely to exist. Indeed, a recent study demonstrated that the Shaker voltage-gated channel activity is modulated by membrane stretch (38)
. Although no physiological function of this modulation is known, it may represent a vestige from a distant ancestor with mechanosensory properties.
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Figure 6. Proposed evolution of the 6TM channels. The cell membrane is represented as two thin parallel lines. Amino- and carboxyl termini of these proteins are both predicted to be intracellular. The third membrane-spanning region (S3) is highlighted in blue. The pore module related domains are colored magenta. TENSIN stands for the tensin-like tyrosin phosphatase domain. The putative ancestral molecules are shown in gray. For the other configurations existing examples are shown: MscL, TPTE, KIR (inward rectifying potassium channels), KcsA (potassium channel from Streptomyces lividans), TPC1 (two-pore channel 1), CatSper (cation channel from sperm), TRP, KChV, NaChV and CaChV, ORK1 (potassium-selective leak channel with two pore domains cloned from Drosophila melanogaster), and TOK1 (outwardly rectifying potassium channel protein with two pore domains arranged in tandem).
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A GLIMPSE OF THE PRIMORDIAL SENSOR?
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Perhaps the most stringent test of functional significance is to find functionality when a domain is expressed alone. Previous studies have demonstrated that S3 synthesized peptides (including the N-h-h-D motif) form an ion pore with many fundamental characteristics of an ion channel when reconstituted into membranes (39)
. The other TMDs, including those normally associated with pore formation, did not. Structural predictions suggest that the ring of conserved aspartates forms the narrowest region of the pore (39)
. Hence, the first primordial channel may have resembled S3.
The number of subunits in the first sensor channel is, of course, speculative. It is remarkable that the Chv channels are tetrameric whereas MscL is pentameric. However, because TMDs are themselves modular, the addition or subtraction of a subunit or a TMD does not require large reconstructions of the protein. An extreme example is the fungal peptide antibiotic ion channel alamethicin, which can contain between 4 and 11 assembled transmembrane helices (40)
.
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CONCLUDING REMARKS
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When combined with those of others, our data suggest the modularity and common evolution of the sensor for a large number of proteins, many of which were not previously placed within the sensor superfamily. Further verification of this hypothesis could be obtained by 1) investigating the TPTE protein for ion conductivity and the voltage dependence of its phosphatase activity; 2) testing whether the prokaryotic sensors identified here encode viable mechano- or voltage sensing channels by assaying for activity; 3) mutating residues of the N-h-h-D motif of additional members of the proposed superfamily; our hypothesis predicts the channel phenotype: loss-of-function if D is mutated, gain-of-function if N; 4) mutate, reconstitute, and assay the activity of channel-forming S3 peptides; the predictions would be similar to full channels (#3 above).
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ACKNOWLEDGMENTS
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The authors would like to thank Drs. Györgyi Szebenyi, Paul Moe, Shmuel Muallem, Ilya Bezprozvanny, Nick Grishin, Kirsten Fischer Lindahl, and Rama Ranganathan for critical reading of the manuscript at its different stages and for their helpful discussions. We give special thanks to Kirsten Fischer Lindahl for her support of this collaboration. The work was funded by American Heart Association grant 9930193N, Robert A Welch Foundation grant I-1420, Air Force Office of Scientific Review grant F49620–01-1–0503, and National Institutes of Health grants GM61028 and DK60818.
Received for publication April 12, 2002.
Accepted for publication June 21, 2002.
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