Cysteine-scanning mutagenesis of transmembrane segments 4 and 5 of the Tn10-encoded metal-tetracycline/H+ antiporter reveals a permeability barrier in the middle of a transmembrane water-filled channel

Cysteine-scanning mutants as to putative transmembrane segments 4 and 5 and the flanking regions of Tn10-encoded metal-tetracycline/H(+) antiporter (TetA(B)) were constructed. All mutants were normally expressed. Among the 57 mutants (L99C to I155C), nine conserved arginine-, aspartate-, and glycine-replaced ones exhibited greatly reduced tetracycline resistance and almost no transport activity, and five conserved glycine- and proline-replaced mutants exhibited greatly reduced tetracycline transport activity in inverted membrane vesicles despite their high or moderate drug resistance. All other cysteine-scanning mutants retained normal drug resistance and normal tetracycline transport activity except for the L142C and I143C mutants. The transmembrane (TM) regions TM4 and TM5 were determined to comprise 20 amino acid residues, Leu-99 to Ile-118, and 17 amino acid residues, Ala-136 to Ala-152, respectively, on the basis of N-[(14)C]ethylmaleimide ([(14)C]NEM) reactivity. The NEM reactivity patterns of the TM4 and TM5 mutants were quite different from each other. TM4 could be divided into two halves, that is, a NEM nonreactive periplasmic half and a periodically reactive cytoplasmic half, indicating that TM4 is tilted toward a water-filled transmembrane channel and that only its cytoplasmic half faces the channel. On the other hand, NEM-reactive mutations were observed periodically (every two residues) along the whole length of TM5. A permeability barrier for a membrane-impermeable sulfhydryl reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, was present in the middle of TM5 between Leu-142 and Gly-145, whereas all the NEM-reactive mutants as to TM4 were not accessible to 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, indicating that the channel-facing side of TM4 is located inside the permeability barrier. Tetracycline protected the G141C mutant from the NEM binding, whereas the other mutants in TM4 and TM5 were not protected by tetracycline.     

Cysteine-scanning mutagenesis around transmembrane segment VI of Tn10-encoded metal-tetracycline/H(+) antiporter

Each amino acid in putative transmembrane helix VI and its flanking regions, from Ser-156 to Thr-185, of a Cys-free mutant of the Tn10-encoded metal-tetracycline/H(+) antiporter (TetA(B)) was individually replaced by Cys. All of the cysteine-scanning mutants showed a normal level of tetracycline resistance except for the S156C mutant, which showed moderate resistance, indicating that there is no essential residue located in this region. All 20 mutants from S159C to W178C showed no reactivity with N-ethylmaleimide (NEM), whereas the mutants of the flanking regions from S156C to H158C and F179C to T185C were highly or moderately reactive with NEM. These results indicate that like transmembrane helices III and IX, the transmembrane helix VI comprising residues Ser-159-Trp-178 is totally embedded in the hydrophobic environment.     

Cysteine-scanning mutagenesis around transmembrane segments 1 and 11 and their flanking loop regions of Tn10-encoded metal-Tetracycline/H+ antiporter

Putative transmembrane helices (TM) 1 and 11 in the metal-tetracycline/H(+) antiporter are predicted to be close to each other on the basis of disulfide cross-linking experiments of the double-cysteine mutants in the periplasmic loop regions (Kubo, Y., Konishi, S., Kawabe, T., Nada, S., and Yamaguchi, A. (2000) J. Biol. Chem. 275, 5270-5274). In this study, each amino acid from Asn-2 to Gly-44 in the putative TM1 and loop1-2 regions or that from Ser-328 to Gly-366 in TM11 and its flanking regions was individually replaced with cysteine. With respect to the TM1 region, 10 mutants, from T5C to L14C, were all not reactive with N-ethylmaleimide (NEM), and from D15C to I22C, NEM-reactive and non-reactive mutations periodically appeared every two residues. Three mutants, M23C to V25C, were all NEM-reactive, but the degree of the latter two mutants was very low. Seven mutants, from L26C to E32C, were all highly reactive with NEM. Therefore, the region of TM1 is composed of the 21 amino acid residues from Thr-5 to Val-25. It is a partially amphiphilic helix, that is, the N-terminal (cytoplasmic) half is embedded in the hydrophobic interior, and the C-terminal (periplasmic) half faces a water-filled channel. With respect to TM11, nine mutants, from S328C to G336C, and six mutants, from L361C to G366C, were all reactive with NEM. On the other hand, out of the 24 mutants, from L337C to S360C, 17 were not reactive with NEM, and the 7 NEM-reactive mutants were scattered, indicating that this region is a transmembrane segment. The 7 residues from Val-347 to Phe-353 including Pro-350 formed a central hydrophobic core, and the 7 NEM-reactive mutations were periodically distributed in its flanking regions, indicating that both ends of TM11 face a water-filled channel. Ala-354 is located at about 1/3 of the length from the periplasmic end of TM11. Disulfide cross-linking experiments on double-cysteine mutants having the combination of A354C and a cysteine-scanning mutation in the loop1-2 region indicated that loop1-2 is very flexible and close to the periplasmic end of TM11. Tetracycline prevented the cross-linking formation between the periplasmic ends of TM1 and TM11; however, it did not affect the cross-linking between loop1-2 and TM11, indicating that the substrate-induced conformational change involves a shift in the relative locations of TM1 and TM11.     

Metal-tetracycline/H+ antiporter of Escherichia coli encoded by a transposon Tn10. Histidine 257 plays an essential role in H+ translocation.

The transposon Tn10-encoded tetA gene product is a metal-tetracycline/proton antiporter (Yamaguchi, A., Udagawa, T., and Sawai, T. (1990) J. Biol. Chem. 265, 4809-4813). Its tetracycline transport activity was inhibited by a histidine-specific reagent, diethyl pyrocarbonate. Among five histidine residues in this antiporter, only His257 is located in the putative transmembrane helices. Thus, His257 was replaced by Glu or Asp. Inverted vesicles containing the Glu257 and Asp257 mutant proteins showed only 20 and 10% of the tetracycline uptake of wild-type vesicles, respectively. In contrast to wild-type vesicles, the mutant vesicles showed no tetracycline-dependent proton translocation, indicating that the mutant proteins had lost the tetracycline/H+ antiport activity. The significant 60Co2+ uptake without proton translocation by the mutant vesicles also confirmed that the mutant carriers act as uniporters of a metal-tetracycline complex. The metal-tetracycline uniport by the mutant proteins was not inhibited by diethyl pyrocarbonate, indicating that His257 is the only histidine residue essential for proton translocation. These mutant proteins conferred about half-level resistance to tetracycline, probably due to their catalyzing downhill efflux of a metal-tetracycline complex out of the cells.     

Metal-tetracycline/H+ antiporter of Escherichia coli encoded by a transposon, Tn10. The role of the conserved dipeptide, Ser65-Asp66, in tetracycline transport

The transposon Tn10-encoded tetracycline resistance protein functions as a metal-tetracycline/H+ antiporter (Yamaguchi, A., Udagawa, T., and Sawai, T. (1990) J. Biol. Chem. 265, 4809-4813). The Ser65-Asp66 dipeptide is conserved in all known tetracycline antiporter proteins and is an important target for site-directed mutagenesis. When Asp66 was replaced by Asn, the transport activity was completely lost, whereas when it was replaced by Glu, the activity was reduced to 10% of the wild-type level, indicating that a negative charge at position 66 is essential for tetracycline transport. Replacement of Ser65 by Cys or Ala, in contrast, caused only a minor change in tetracycline transport activity. However, the Cys65 mutant antiporter was sensitive to sulfhydryl reagents. Complete inactivation of the Cys65 antiporter by N-ethylmaleimide was not prevented by the substrate. A less bulky reagent, methyl methanethiosulfonate, caused partial inactivation of the Cys65 antiporter without changing its affinity to the substrate. These results indicate that a region including the dipeptide plays an important role in metal-tetracycline transport except for substrate binding. It may act as a gate which opens on the charge-charge interaction between Asp66 and the metal-tetracycline.     

The fourth transmembrane domain of the Helicobacter pylori Na+/H+ antiporter NhaA faces a water-filled channel required for ion transport

Cysteine-scanning mutagenesis was performed from Ser-130 to Leu-160 in the fourth transmembrane domain (TM4) of the Na+/H+ antiporter NhaA from Helicobacter pylori to determine the topology of each residue and to identify functionally important residues. All of the mutants were based on cysteine-less NhaA (Cys-less NhaA), which functions very similarly to the wild-type protein, and were expressed at a level similar to Cys-less NhaA. Discontinuity of [14C]N-ethylmaleimide (NEM)-reactive residues suggested that TM4 comprises residues Gly-135 to Val-156. Even within TM4, NEM reactivity was high for I136C, D141C to A143C, L146C, M150C, and G153C to R155C. These residues are thought to be located on one side of the -helical structure of TM4 and to face a putative water-filled channel. Pretreatment of intact cells with membrane-impermeable maleimide did not inhibit [14C]NEM binding to the NEM-reactive residues within TM4, suggesting that the putative channel opens toward the cytoplasm. NEM reactivity of the A143C mutant was significantly inhibited by Li+. The T140C and D141C mutants showed lower affinity for Na+ and Li+ as transport substrates, but their maximal antiporter velocities (Vmax) were relatively unaffected. Whereas the I142C and F144C mutants completely lost their Li+/H+ antiporter activity, I142C had a lower Vmax for the Na+/H+ antiporter. F144C exhibited a markedly lower Vmax and a partially reduced affinity for Na+. These results suggest that Thr-140, Asp-141, and Phe-144 are located in the end portion of a putative water-filled channel and may provide the binding site for Na+, Li+, and/or H+. Furthermore, residues Ile-142 to Phe-144 may be important for the conformational change that accompanies ion transport in NhaA.     

Transmembrane remote conformational suppression of the Gly-332 mutation of the Tn10-encoded metal-tetracycline/H+ antiporter.

Gly-332 is a conformationally important residue of the Tn10-encoded metal-tetracycline/H+ antiporter (TetA(B)), which was found by random mutagenesis and confirmed by site-directed mutagenesis. A bulky side chain at position 332 is deleterious to the transport function. A spontaneous second-site suppressor revertant was isolated from G332S mutant and identified as the Ala-354-->Asp mutant. Gly-332 and Ala-354 are located on opposite ends of transmembrane segment XI. As judged from [14C]NEM binding to Cys mutants, the residue at position 354, which is originally exposed to water, was buried in the membrane by a G332S mutation through a remote conformational change of transmembrane segment XI. This effect is the same as that of a G62L mutation at position 30 through transmembrane segment II [Kimura, T., Sawai, T. and Yamaguchi, A. (1997) Biochemistry 36, 6941-6946]. Interestingly, the G332S mutation was also suppressed by the L30S mutation, and the G62L mutation was moderately suppressed by the A354D mutation. These results indicate the presence of a close conformational relationship between the flanking regions of the transmembrane segments II and XI.     

Metal-tetracycline/H+ antiporter of Escherichia coli encoded by transposon Tn10. Roles of the aspartyl residues located in the putative transmembrane helices.

Three conserved aspartyl residues located in the putative transmembrane helices in the Tn10-encoded metal-tetracycline/H+ antiporter were replaced by Asn, Lys, or Glu with oligonucleotide-directed site-specific mutagenesis. Replacement of Asp84 or Asp15 by Asn or Lys caused a severe defect in tetracycline transport activity, however, the Glu84 and Glu15 mutants retained 150 and 40% of the wild type activity, respectively, indicating the critical role of the negative charge. The increase in the activity of the Glu84 mutant was due to an increase in the affinity for the substrate. H+/tetracycline coupling was intact in these mutants, including Asn and Lys mutants. On the other hand, all of the Asp285-substitution mutants showed a severe defect in tetracycline transport activity and a complete lack of tetracycline-coupled H+ transport. However, since in vivo tests showed the tetracycline resistance for the Glu285 mutant, a negative charge in position 285 plays some role in maintaining the possible down-hill and/or low affinity efflux of accumulated tetracycline from intact cells. Similar work was done for Asp365, and here the Asn and Glu mutants showed decreased but high activity, while the Lys mutant was only marginally active (5%), indicating that a negative charge is not so demanding in position 365, possibly because it is not in the membrane.     

Serine residues responsible for tetracycline transport are on a vertical stripe including Asp-84 on one side of transmembrane helix 3 in transposon Tn10-encoded tetracycline/H+ antiporter of Escherichia coli.

Putative transmembrane helix 3 of the tetracycline/H+ antiporter encoded by a transposon, Tn10, contains four serine residues, Ser-77, Ser-82, Ser-91 and Ser-92. Each of these serine residues was replaced by site-directed mutagenesis. Of these four serine residues, Ser-77 was important for the transport function, and a bulky side chain at position 91 hindered substrate translocation, whereas Ser-82 and Ser-92 did not play any role. Ser-77 and Ser-91 are on the same vertical stripe, that includes the essential Asp-84, on the hydrophilic side of putative helix 3. These observations suggest that helix 3 is part of the tetracycline translocation channel across the membrane.     

Ion binding affinity in the cavity of the KcsA potassium channel

The hydrophobic cell membrane interior presents a large energy barrier for ions to permeate. Potassium channels reduce this barrier by creating a water-filled cavity at the middle of their ion conduction pore to allow ion hydration and by directing the C-terminal "end charge" of four alpha-helices toward the water-filled cavity. Here we have studied the interaction of monovalent cations with the cavity of the KcsA K(+) channel using X-ray crystallography. In these studies, Tl(+) was used as an analogue for K(+) and the total ion-stabilization energy for Tl(+) in the cavity was estimated by measuring its binding affinity. Binding affinity for the Na(+) ion was also measured, revealing a weak selectivity ( approximately 7-fold) favoring Tl(+) over Na(+). The structures of the cavity containing Na(+), K(+), Tl(+), Rb(+), and Cs(+) are compared. These results are consistent with a fairly large (more negative than -100 mV) electrostatic potential inside the cavity, and they also imply the presence of a weak nonelectrostatic component to a cation's interaction with the cavity.     

Aspartic acid-66 is the only essential negatively charged residue in the putative hydrophilic loop region of the metal-tetracycline/H+ antiporter encoded by transposon Tn10 of Escherichia coli.

Of the 16 acidic amino acid residues located in the hydrophilic region of the metal-tetracycline/H+ antiporter of transposon Tn10, five glutamic acids and three aspartic acids are conserved among the tetracycline/H+ antiporters of Gram-negative bacteria. When these conserved acidic residues were each replaced by a neutral polar residue, glutamine or asparagine, only the Asp66 substitution mutants completely lost their transport activity. The substitution of Glu274, Asp120, Glu181, or Asp38 caused significant reduction of the transport activity, whereas the substitution of the other three residues had no detectable effect on the activity. These findings led to the conclusion that only Asp66 is essential for the transport function.     

Engineered Tet repressor mutants with single tryptophan residues as fluorescent probes. Solvent accessibilities of DNA and inducer binding sites and interaction with tetracycline

Mutants of the Tn10-encoded Tet repressor containing single or no tryptophan residues were constructed by oligonucleotide-directed mutagenesis. The Trp-75 to Phe exchange reduces the dissociation rate of the complex with the inducer tetracycline by a factor of 2. The Trp-43 to Phe exchange has no effect on inducer binding. The fluorescence emission spectra of both tryptophan residues are quenched to a different extent by binding of tetracycline: Trp-75 is quenched to zero and Trp-43 to only 50%. It is concluded that Trp-75 is in the vicinity of the inducer binding site. The different fluorescence emission spectra of both tryptophan residues depend on the native structure of Tet repressor. Quenching studies with iodide indicate that the DNA binding motif is solvent exposed in free repressor and moves towards the interior of the protein upon inducer binding. The inducer binding site is in the interior of the protein. The fluorescence of tetracycline is enhanced upon binding to Tet repressor. The excitation at 280 nm results mainly from the change in environment and in part from energy transfer from tryptophan to the drug.     

Role of the charge interaction between Arg(70) and Asp(120) in the Tn10-encoded metal-tetracycline/H(+) antiporter of Escherichia coli

We reported that the positive charge of Arg(70) is mandatory for tetracycline transport activity of Tn10-encoded metal-tetracycline/H(+) antiporter (TetA(B)) (Someya, Y., and Yamaguchi, A. (1996) Biochemistry 35, 9385-9391). Arg(70) may function through a charge-pairing with a negatively charged residue in close proximity. Therefore, we mutated Asp(66) and Asp(120), which are only two negatively charged residues located close to Arg(70) in putative secondary structure of TetA(B) and highly conserved throughout transporters of the major facilitator superfamily. Site-directed mutagenesis studies revealed that Asp(66) is essential, but Asp(120) is important for TetA(B) function. Surprisingly, when Asp(120) was replaced by a neutral residue, the R70A mutant recovered tetracycline resistance and transport activity. There was no such effect in the Asp(66) mutation. The charge-exchanged mutant, R70D/D120R, also showed significant drug resistance and transport activity (about 50% of the wild type), although the R70D mutant had absolutely no activity, and the D120R mutant retained very low activity (about 10% of the wild type). Both the R70C and D120C mutants were inactivated by N-ethylmaleimide. Mercuric ion (Hg(2+)), which gives a positive charge to a SH group of a Cys residue through mercaptide formation, had an opposite effect on the R70C and D120C mutants. The activity of the R70C mutant was stimulated by Hg(2+); however, on the contrary, the D120C mutant was partially inhibited. On the other hand, the R70C/D120C double mutant was almost completely inactivated by Hg(2+), probably because the side chains at positions 70 and 120 are bridged with Hg(2+). The close proximity of positions 70 and 120 were confirmed by disulfide cross-linking formation of the R70C/D120C double mutant when it was oxidized by copper-(1,10-phenanthroline). These results indicate that the positive charge of Arg(70) requires the negative charge of Asp(120) for neutralization, probably for properly positioning transmembrane segments in the membrane.     

Structural conservation in the major facilitator superfamily as revealed by comparative modeling

The structures of membrane transporters are still mostly unsolved. Only recently, the first two high-resolution structures of transporters of the major facilitator superfamily (MFS) were published. Despite the low sequence similarity of the two proteins involved, lactose permease and glycerol-3-phosphate transporter, the reported structures are highly similar. This leads to the hypothesis that all members of the MFS share a similar structure, regardless of their low sequence identity. To test this hypothesis, we generated models of two other members of the MFS, the Tn10-encoded metal-tetracycline/H(+) antiporter (TetAB) and the rat vesicular monoamine transporter (rVMAT2). The models are based on the two MFS structures and on experimental data. The models for both proteins are in good agreement with the data available and support the notion of a shared fold for all MFS proteins.     

Structure-function relationship of the fifth transmembrane domain in the Na+/H+ antiporter of Helicobacter pylori: Topology and function of the residues, including two consecutive essential aspartate residues

We examined the structure-function relationships of residues in the fifth transmembrane domain (TM5) of the Na+/H+ antiporter A (NhaA) from Helicobacter pylori (HP NhaA) by cysteine scanning mutagenesis. TM5 contains two aspartate residues, Asp-171 and Asp-172, which are essential for antiporter activity. Thirty-five residues spanning the putative TM5 and adjacent loop regions were replaced by cysteines. Cysteines replacing Val-162, Ile-165, and Asp-172 were labeled with NEM, suggesting that these three residues are exposed to a hydrophilic cavity within the membrane. Other residues in the putative TM domain, including Asp-171, were not labeled. Inhibition of NEM labeling by the membrane impermeable reagent AMS suggests that Val-162 and Ile-165 are exposed to a water filled channel open to the cytoplasmic space, whereas Asp-172 is exposed to the periplasmic space. D171C and D172C mutants completely lost Na+/H+ and Li+/H+ antiporter activities, whereas other Cys replacements did not result in a significant loss of these activities. These results suggest that Asp-171 and Asp-172 and the surrounding residues of TM5 provide an essential structure for H+ binding and Na+ or Li+ exchange. A168C and Y183C showed markedly decreased antiporter activities at acidic pH, whereas their activities were higher at alkaline pH, suggesting that the conformation of TM5 also plays a crucial role in the HP NhaA-specific acidic pH antiporter activity.     

Cysteine scanning mutagenesis of helices 2 and 7 in GLUT1 identifies an exofacial cleft in both transmembrane segments

Cysteine scanning mutagenesis in conjunction with site-directed chemical modification of sulfhydryl groups by p-chloromercuribenzenesulfonate (pCMBS) or N-ethylmaleimide (NEM) was applied to putative transmembrane segments (TM) 2 and 7 of the cysteine-less glucose transporter GLUT1. Valid for both helices, the majority of cysteine substitution mutants functioned as active glucose transporters. The residues F72, G75, G76, G79, and S80 within helix 2 and G286 and N288 within helix 7 were irreplaceable because the mutant transporters displayed transport activities that were lower than 10% of Cys-less GLUT1. The indicated cluster of glycine residues within TM 2 is located on one face of the helix and may provide space for a bulky hydrophobic counterpart interacting with another transmembrane segment or lipid side chains. Characteristic for helix 7, three glutamine residues (Q279, Q282, and Q283) played an important role in transport activity of Cys-less GLUT1 because an individual replacement with cysteine reduced their transport rates by about 80%. ParaCMBS-sensitivity scanning of both transmembrane segments detected several membrane-harbored residues to be accessible to the extracellular aqueous solvent. The pCMBS-reactive sulfhydryl groups were located exclusively in the exofacial half of the plasma membrane and, when presented in a helical model, lie along one side of the helices. Taken together, transmembrane segments 2 and 7 form clefts accessible to the extracellular aqueous solvent. The lining residues are however excluded from interaction with intracellular solutes, as justified by microinjection of pCMBS into the cytoplasm of Xenopus oocytes.     

Site-directed spin-labeling analysis of reconstituted Mscl in the closed state

The mechanosensitive channel from Escherichia coli (Eco-MscL) responds to membrane lateral tension by opening a large, water-filled pore that serves as an osmotic safety valve. In an attempt to understand the structural dynamics of MscL in the closed state and under physiological conditions, we have performed a systematic site-directed spin labeling study of this channel reconstituted in a membrane bilayer. Structural information was derived from an analysis of probe mobility, residue accessibility to O(2) or NiEdda and overall intersubunit proximity. For the majority of the residues studied, mobility and accessibility data showed a remarkable agreement with the Mycobacterium tuberculosis crystal structure, clearly identifying residues facing the large water-filled vestibule at the extracellular face of the molecule, the narrowest point along the permeation pathway (residues 21-26 of Eco-MscL), and the lipid-exposed residues in the peripheral transmembrane segments (TM2). Overall, the present dataset demonstrates that the transmembrane regions of the MscL crystal structure (obtained in detergent and at low pH) are, in general, an accurate representation of its structure in a membrane bilayer under physiological conditions. However, significant differences between the EPR data and the crystal structure were found toward the COOH-terminal end of TM2.     

An energy-efficient gating mechanism in the acetylcholine receptor channel suggested by molecular and Brownian dynamics

Acetylcholine receptors mediate electrical signaling between nerve and muscle by opening and closing a transmembrane ion conductive pore. Molecular and Brownian dynamics simulations are used to shed light on the location and mechanism of the channel gate. Four separate 5 ns molecular dynamics simulations are carried out on the imaged structure of the channel, a hypothetical open structure with a slightly wider pore and a mutant structure in which a central ring of hydrophobic residues is replaced by polar groups. Water is found to partially evacuate the pore during molecular simulations of the imaged structure, whereas ions face a large energy barrier and do not conduct through the channel in Brownian dynamics simulations. The pore appears to be in a closed configuration despite containing an unobstructed pathway across the membrane as a series of hydrophobic residues in the center of the channel provide an unfavorable home to water and ions. When the channel is widened slightly, water floods into the channel and ions conduct at a rate comparable to the currents measured experimentally in open channels. The pore remains permeable to ions provided the extracellular end of the pore-lining helix is restrained near the putative open configuration to mimic the presence of the ligand binding domain. Replacing some of the hydrophobic residues with polar ones decreases the barrier for ion permeation but does not result in significant currents. The channel is posited to utilize an energy efficient gating mechanism in which only minor conformational changes of the hydrophobic region of the pore are required to create macroscopic changes in conductance.     

Mutational and sequence analysis of transmembrane segment 6 orientation in TetA proteins

The packing orientations of the 8 transmembrane (TM) segments that line the central, aqueous transport channel within tetracycline resistance proteins (TetA) have been established. However, the orientations of the remaining 4 segments, TMs 3, 6, 9, and 12, located at the periphery, and away from the transport channel, have not yet been determined. In this study, the packing orientation of TM6 within the class C TetA protein encoded by plasmid pBR322 was evaluated by substitution mutagenesis and analysis of sequence conservation and amphipathicity. The combined data support a model in which the conserved and polar face of the TM6 alpha-helix containing Asn170 and Asn173 orients towards channel-lining TM segments, and the relatively non-conserved and hydrophobic face of TM6 points towards membrane lipids.     

Homology model of dihydropyridine receptor: implications for L-type Ca(2+) channel modulation by agonists and antagonists

L-type calcium channels (LCCs) are transmembrane (TM) proteins that respond to membrane depolarization by selectively permeating Ca(2+) ions. Dihydropyridine (DHP) agonists and antagonist modulate Ca(2+) permeation by stabilizing, respectively, the open and closed states of the channel. The mechanism of action of these drugs remains unclear. Using, as a template, the crystal structure of the KcsA K(+) channel (Doyle et al. (1998) Science 280, 69-77), we have built several homology models of LCC with alternative alignments of TM segments between the proteins. In each model, nifedipine was docked in the pore region and in the interface between repeats III and IV. Several starting structures were generated by constraining the ligand to residues whose mutations reportedly affect DHP binding (DHP-sensing residues). These structures were Monte Carlo-minimized with and without constraints. In the complex with the maximum number of contacts between the ligand and DHP-sensing residues and the lowest ligand-receptor energy, the drug fits snugly in the "water-lake" cavity between segments S6s, which were aligned with M2 segment of KcsA as proposed for Na(+) channel (Lipkind and Fozzard (2000) Biochemistry 39, 8161-8170). In the flattened-boat conformation of DHP ring, the NH group at the stern approaches the DHP-sensing tyrosines in segments IIIS6 and IVS6. Stacking interactions of IVS6 Tyr with the bowsprit aromatic ring stabilize the ligand's orientation in which the starboard COOMe group coordinates Ca(2+) ion chelated by two conserved glutamates in the selectivity filter. In the inverted teepee structure of LCC, the portside COOMe group approaches a bracelet of conserved hydrophobic residues at the helical-bundle crossing, which may function as the activation gate. The dimensions of the gate may readily change upon small rotation of the pore-forming TM segments. The end of the portside group is hydrophobic in nifedipine, (R)-Bay K 8644, and other antagonists. Favorable interactions of this group with the hydrophobic bracelet would stabilize its closed conformation. In contrast, (S)-Bay K 8644 and several other agonists have hydrophilic groups at the portside. Unfavorable interactions of the hydrophilic group with the hydrophobic bracelet would destabilize its closed conformation thereby stabilizing the open conformation. In the agonist-bound channel, Ca(2+) ions would permeate between the hydrophilic face of the ligand and conserved hydrophilic residues in segments IS6 and IIS6. Our model suggests mutational experiments that could further our understanding of the pharmacological modulation of voltage-gated ion channels.