Conversion of the Ion Selectivity of the 5-HT3A Receptor from Cationic to Anionic Reveals a Conserved Feature of the Ligand-gated Ion Channel Superfamily*

Martin J. GunthorpeDagger § and Sarah C. R. LummisDagger ||

From the Dagger  Division of Neurobiology, Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom and the  Department of Biochemistry, Tennis Court Road, Cambridge CB2 1QW, United Kingdom

Received for publication, October 19, 2000, and in revised form, December 26, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

The 5-hydroxytryptamine3 (5-HT3) receptor is a member of a superfamily of ligand-gated ion channels, which includes nicotinic acetylcholine, gamma -aminobutyric acid, and glycine receptors. The receptors are either cation or anion selective, leading to their distinctive involvement in either excitatory or inhibitory neurotransmission. Using a combination of site-directed mutagenesis and electrophysiological characterization of homomeric 5-HT3A receptors expressed in HEK293 cells, we have identified a set of mutations that convert the ion selectivity of the 5-HT3A receptor from cationic to anionic; these were substitution of V13'T in M2 together with neutralization of glutamate residues (E-1'A) and the adjacent insertion of a proline residue (P-1') in the M1-M2 loop. Mutant receptors showed significant chloride permeability (PCl/PNa = 12.3, PNa/PCl = 0.08), whereas WT receptors are predominantly permeable to sodium (PNa/PCl > 20, PCl/PNa < 0.05). Since the equivalent mutations have previously been shown to convert alpha 7 nicotinic acetylcholine receptors from cationic to anionic (Galzi J.-L., Devillers-Thiery, A, Hussy, N., Bertrand, S. Changeux, J. P., and Bertrand, D. (1992) Nature 359, 500-505) and, recently, the converse mutations have allowed the construction of a cation selective glycine receptor (Keramidas, A., Moorhouse, A. J., French, C. R., Schofield, P. R., and Barry, P. H. (2000) Biophys. J. 78, 247-259), it appears that the determinants of ion selectivity represent a conserved feature of the ligand-gated ion channel superfamily.



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ABSTRACT
INTRODUCTION
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5-HT31 receptors are ligand-gated ion channels (LGICs) whose activation results in membrane depolarization due to the gating of an integral cation-selective channel (3, 4). 5-HT3 receptors can exist as homomeric assemblies of 5-HT3A receptor subunits or heteromeric assemblies of 5-HT3A and 5-HT3B receptor subunits; these have distinct physical properties (5, 6). 5-HT3 receptors form part of a phylogenetically linked superfamily of LGIC, which is typified by the nicotinic acetylcholine (nACh) receptor, also includes the glycine and gamma -aminobutyric acid type A receptors and is thought to have arisen from a common ancestor over 2000 million years ago (7). All of these receptors are believed to be constructed from a pseudopentameric arrangement of subunits around a central water-filled pore. Individual subunits are predicted to contain a large N-terminal domain and four transmembrane spanning domains (M1-M4), arranged such that M2 (see Fig. 1 for residue numbering and lettering in MZ) delineates the wall of the channel. Agonist binding to the N-terminal domain results in a conformational change that is communicated to the pore domain, resulting in the opening of the channel. Evidence suggests that as well as the conservation of structural features, the underlying molecular mechanisms of operation of these receptors are also conserved (8, 9). However, once the channels have opened, one crucial difference becomes clear; whereas the integral ion channels of the 5-HT3 and nACh receptors are cation-selective and are primarily concerned with excitatory neurotransmission, glycine and gamma -aminobutyric acid receptors are anion-selective and are consequently involved in inhibitory neurotransmission.

The occurrence of both anionic and cationic-selective receptors in such a closely related family provides a basis for the identification of the molecular determinants of ion selectivity. Since there is overwhelming evidence to support a major role of the M2 domain in forming the walls of the ion channel (for a review, see Ref. 10), previous studies have concentrated on this region of the receptor in the search for amino acids that play a role in ion conduction. For instance, site-directed mutagenesis studies on acetylcholine receptors have identified rings of residues that alter channel gating (11), conductance (12), or the selectivity among monovalent (12, 13) or divalent (14) cations. Alignment of the primary amino acid sequences of the M2 regions of the different members of the LGIC superfamily (a number of representative sequences are shown in Fig. 1) highlights the striking levels of conservation found between the M2 domains of both the cationic and anionic receptors. There are, however, noticeable differences, and it is an evaluation of the differences between the cationic alpha 7 nACh and anionic glycine receptor M2 regions that allowed Galzi et al. (1) to identify the determinants of cation versus anion selectivity for the alpha 7 nACh receptor. Initially, they incorporated all of the alpha 1 glycine receptor residues presumed to face the channel lumen into the alpha 7 nACh receptor and demonstrated that this resulted in an anionic alpha 7 nACh receptor. Subsequently, the number of point mutations was reduced until the smallest subset of determinants of the cationic versus anionic selectivity were obtained. Three mutations were found to be essential: the substitution of V13'T together with the neutralization of a ring of glutamate residues (E-1'A) and the adjacent insertion of the "extra" amino acid (proline) found in the anionic channels (referred to here as P-1') in the M1-M2 loop. Subsequently, the "reverse" mutations in the alpha 1 glycine receptor have been shown to change the ionic selectivity of this receptor (2). A schematic representation of these three rings of residues in the channel domain of the receptor is shown in Fig. 1B.

To investigate whether the same structural features are important in determining the ion selectivity of the 5-HT3 receptor, we introduced the equivalent mutations into the murine 5-HT3A receptor subunit (Fig. 1), which was then expressed in HEK293 cells and characterized using the whole-cell configuration of the patch clamp technique. Our results demonstrate that the mutant 5-HT3A receptor exhibits a clear anion selectivity.


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Cell Culture and Transfection-- Human embryonic kidney (HEK 293) cells stably expressing 5-HT3A receptors were developed using the eukaryotic expression vector pRc/CMV (InVitrogen, Abingdon, UK) containing the complete coding sequence for the 5-HT3A(b) subunit from NIE-115 cells as previously described (16). The 5-HT3A-STM, which incorporates three point mutations, E-1'A, the insertion of a proline between -1' and -2' positions (referred to as -1' in this study), and V13'T in the M2 region of the 5-HT3 receptor, was generated by two successive rounds of oligonucleotide-directed mutagenesis using the Kunkel method (17) and confirmed by DNA sequencing. The oligonucleotides used in the reactions were as follows: V13'T, GATGATGAGGAAAGTACTGTATCCCAGAAG; P-1'/E-1'A: GAGACTCTCGCGGGCCCACTGTCCG. Cell lines were routinely grown on 90-mm tissue culture plates in a 1:1 mix of Dulbecco's modified Eagle's medium and F-12 supplemented with 10% fetal calf serum and 500 µg/ml geneticin in a humidified atmosphere at 37 °C and 7% CO2. For transient transfections, HEK 293 cells at 60-70% confluency (~48 h postpassage) were transfected with WT or mutant plasmid DNA by the calcium phosphate precipitation method (18). For electrophysiology, experiments cells were grown on 35-mm plates (Falcon), and recordings were performed on cells 1-4 days post-transfection.

Electrophysiological Procedures-- In the majority of experiments, whole-cell currents from single cells were recorded using an EPC-9 amplifier in conjunction with HEKA software as described previously (19); however, for voltage ramp experiments, an Axopatch 200B amplifier (Axon Instruments Inc.) controlled using the pClamp7 software suite was used. In most experiments, cells were perfused with an extracellular solution (E1) of the following composition: 130 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1.1 mM EGTA, 2 mM MgCl2, 10 mM HEPES, 30 mM glucose, pH 7.2, with NaOH. Patch pipettes were made from thin walled borosilicate glass capillary tubing (Clark Electromedical GC120F-10) and filled with intracellular solution (I1; 140 mM CsCl, 1 mM MgCl2, 0.1 mM CaCl2, 10 mM HEPES titrated to pH 7.2 with CsOH). Electrode resistances measured with these solutions were typically 2-5 megaohms. In ion selectivity experiments, solutions with modified ionic compositions were used. In our first series of experiments, the extracellular NaCl concentration was reduced, and the osmolarity of the resulting solution was maintained by the addition of mannitol (E2; as E1, except 65 mM NaCl, 130 mM mannitol). The extracellular chloride concentration was also reduced by substituting chloride with the anion isethionate (E3; as E1 except 15 mM NaCl, 115 mM sodium isethionate). In further experiments designed to allow the accurate determination of permeability ratios (PCl/PNa or PNa/PCl), we used simplified versions of the intracellular and extracellular solutions above, employing Na+ as the predominant cation. The sodium-based intracellular solution (I2) contained 140 mM NaCl, 0.1 mM MgCl2, 0.1 mM CaCl2, 1.1 mM EGTA, 10 mM HEPES titrated to pH 7.2 with 5 mM NaOH, and the simplified extracellular solution (E4) used contained 140 mM NaCl, 0.1 mM MgCl2, 1 mM CaCl2, 30 mM glucose, 10 mM HEPES titrated to pH 7.2 with 5 mM NaOH. Again, an extracellular solution (E5) involving isethionate substitution for chloride was employed (as E4 except 126 mM sodium isethionate, 14 mM NaCl). Liquid junction potentials arising at the tip of the patch pipette were calculated by the method of Barry and Lynch (20), and potential measurements were corrected post hoc. Drugs were applied in one of three ways: via a U-tube device capable of complete solution exchange within 100 ms (19), via a DAD-12 perfusion system (ALA Scientific Instruments Inc.) (21), or, for voltage ramp experiments, via a Warner Instruments SF-77B fast solution exchange system (22), both of which are capable of solution exchange in ~30 ms. Current-voltage relationships were established by measuring the peak current evoked by 10 µM 5-HT at holding potentials between -80 and +40 mV or by the application of voltage ramps (-70 to +100 mV, in 200 ms) during the plateau phase of an agonist-evoked response. Dose-response data were fitted with a logistic function using Kaleidagraph (Abelbeck Software) according to the equation I = Imax/(1 + (EC50/[L])n), where Imax is the maximal response, EC50 is the concentration of agonist required for half-maximal effect, [L] is the concentration of agonist, and n is the Hill coefficient. Permeability ratios for Na+ and Cl- were calculated according to the Goldman-Hodgkin-Katz (voltage) equation (23): Vrev = (RT/F)ln((PNa[Na]o + PCl[Cl]i)/(PNa[Na]i PCl[Cl]o)), where R, T, and F have their usual meaning, Vrev is the reversal potential obtained from the current-voltage relationship, Pion is the permeability of the ion, and the subscripts i and o refer to intracellular and extracellular ion concentrations, respectively. Data were analyzed using Kaleidagraph, Origin (MicroCal), or Clampex (Axon Instruments) software, and all data are quoted as the mean ± S.E. for n independent experiments. Where appropriate, Student's t test for unpaired data was used, and values of p < 0.05 were regarded as significant.

Drugs and Reagents-- All cell culture reagents were from Life Technologies, Inc. except fetal calf serum, which was from Sigma. 5-HT hydrochloride was from Research Biochemicals Inc. (St. Albans, UK). All other reagents were from Sigma.


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Functional Characterization of WT and Mutant 5-HT3 Receptors Expressed in HEK293 Cells-- The mutant 5-HT3A receptor, which we have called the 5-HT3A-STM (for selectivity triple mutation), containing the three point mutations defined in Fig. 1 was functionally expressed in HEK293 cells, giving rise to 5-HT-gated whole-cell currents, which were completely blocked by coapplication with the selective 5-HT3 receptor antagonist granisetron (100 nM; Fig. 2A). Dose-response curves revealed that the 5-HT3A-STM had a 10-fold lower EC50 for 5-HT (240 ± 30 nM, n = 5) compared with the WT receptor (Fig. 2C; Table I) (19) with a reduced Hill coefficient but no significant difference in the maximum amplitude of the responses (Imax) (Table I). The 5-HT3A-STM receptor showed markedly different activation and desensitization kinetics compared with the WT receptor. In response to a maximal concentration of 5-HT, 5-HT3A-STM receptor whole-cell currents peaked within 684 ± 117 ms (10-90% rise time 316 ± 59 ms, n = 8), significantly more slowly than for the WT receptor (time to peak 198 ± 14 ms, 10-90% rise time 103 ± 9 ms, n = 11; see Fig. 3 and Table I). Furthermore, while both exhibited desensitization in the continued presence of a maximal concentration of agonist (30 µM 5-HT), the rate of desensitization was clearly much slower for 5-HT3A-STM. The time course of agonist-induced desensitization could be fitted with a single exponential, giving a time constant of 44.7 ± 3.6 s (n = 3) for the 5-HT3A-STM, more than 10-fold slower than the WT receptor (3.4 ± 0.5 s, n = 5; Table I). Surprisingly, shorter duration applications of agonist (30 µM 5-HT, 5 s) to cells expressing the 5-HT3A-STM receptor produced whole-cell responses with a different time course of recovery; receptors appeared to remain in a conducting state for >20 s after agonist removal (see Fig. 3). This combination of slower activation, reduced desensitization, and increased affinity exhibited by the 5-HT3A-STM suggests that this receptor may attain a desensitized but conducting state (D*) in the presence of agonist, similar to that identified in some mutant acetylcholine receptors (1, 24, 25). Some of these receptors show spontaneous activity (channel opening), and some are gated by antagonists (1, 26). We therefore examined the effect of the application of the 5-HT3 receptor antagonists 100 nM granisetron or 100 nM D-tubocurarine alone to the 5-HT3A-STM receptor. Both antagonists had no apparent effect; they did not reduce leak currents (which would be indicative of spontaneous channel activity) or cause activation of 5-HT3A-STM receptors (data not shown). Experiments to examine the single channel conductance of the WT and 5-HT3A-STM receptors and hence address the possibility of the occurrence of a secondary D* state were hampered by the low single channel conductance of recombinant 5-HT3A receptors (Ref. 19; see "Discussion") and were not pursued further.



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Fig. 1.   Primary amino acid sequences of channel lining regions of members of the ligand-gated ion channel superfamily. A, an alignment of the channel lining regions of representative amino acid sequences of the 5-HT3A receptor subunit and those from related LGIC are shown. The region illustrated includes the M2 transmembrane domain with individual amino acids being referred to by the single letter code devised by Miller (41). Boxes indicate amino acids that are highly conserved among cationic and anionic receptors. The arrows indicate amino acids that differ between anionic and cationic receptors and that have been mutated in this study. The 5-HT3A-STM mutant illustrated contains the E-1'A and V13'T point mutations and the insertion of a proline residue between G-2' and E-1', which is referred to as P-1" in this study. The position of the cytoplasmic (-4'), intermediate (-1'), and extracellular (20') rings of charged residues bordering M2 in the cationic channels (12) are also indicated. B, a schematic representation of the channel domain of a LGIC showing the rings of amino acids that have been mutated in this study.



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Fig. 2.   Characterization of 5-HT3A-STM receptors expressed in HEK 293 cells. A, 5-HT3A-STM receptor currents were reversibly antagonized by the selective 5-HT3 receptor antagonist granisetron (100 nM). The antagonist was preapplied for 20 s before being coapplied with 5-HT and had no effect alone (data not shown). B, typical whole-cell currents from experiments used to establish the dose-response relationship for 5-HT3A-STM receptor activation by 5-HT. C, graph illustrating the pooled, normalized, concentration response data determined from experiments similar to those illustrated in A. The parameters determined from curve fits according to the Hill equation are given in Table I. All traces shown are from single experiments but are representative of at least three others


                              
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Table I
Activation and desensitization properties of WT and 5-HT3A-STM receptors
Data shown are the mean ± S.E. (n) shown in parentheses.



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Fig. 3.   Activation and desensitization of WT and 5-HT3A-STM receptors. A-C, example of whole-cell current traces of 5-HT-gated currents in HEK 293 cells stably expressing WT or 5-HT3A-STM receptors. Currents were evoked by the application of 30 µM 5-HT for the duration indicated by the solid bars. 5-HT3A-STM responses were slower to activate than WT receptors and desensitized more slowly in the continuous presence of agonist (see Table I). Shorter duration applications of 5-HT resulted in a different time course of recovery of 5-HT3A-STM receptor currents. The vertical calibration bar corresponds to 200 pA. D, the activation time course for WT and 5-HT3A-STM receptors is shown on an expanded time scale. E, the differences in the time course of recovery of the 5-HT3A-STM receptor due to different agonist application durations are highlighted by overlay of the traces shown in B and C. All traces shown are from single experiments but are representative of at least three others.

Ion Selectivity of the WT and 5-HT3A-STM Receptors-- To compare the ion selectivity of the WT and mutant receptors, we initially constructed current-voltage relationships in a number of different extracellular solutions, E1 (normal extracellular solution), E2 (NaCl-mannitol), and E3 (NaCl-isethionate), which differ in their Na+ and/or Cl- concentrations. Sample traces illustrating the WT and 5-HT3A-STM receptor currents recorded at the range of holding potentials studied to allow construction of a steady state current-voltage relationship are shown (Fig. 4, A and B). The resulting current-voltage relationships obtained from such data sets are given in Fig. 4C, and the reversal potentials estimated from the current-voltage data are given in Table II. The reversal potential of the WT receptor in normal extracellular solution (E1) was -9.0 ± 1.6 mV. The data show that reduction of the extracellular NaCl concentration causes a leftward shift in the reversal potential from -9.0 ± 1.6 mV to -19.5 ± 2.0 mV (Delta Vrev = -10.5 mV), indicative of a cation-selective channel. The reversal potential of the WT receptor in NaCl-isethionate solution (E3) was -7.1 ± 1.3 mV. This value is not significantly different from that determined in solution E1 (Delta Vrev ~ 0 mV) and indicates that chloride ions make no significant contribution to the ion current passing through the WT receptor. In the equivalent I-V experiments conducted for the 5-HT3A-STM receptor, the reversal potential in normal extracellular solution (E1) was 0.1 ± 1.8 mV, and reduction of the extracellular NaCl concentration caused a rightward shift in the reversal potential to 21.8 ± 0.9 mV (Delta Vrev = +21.7 mV). Reduction of the extracellular Cl- concentration also caused a rightward shift in the reversal potential from 0.1 ± 1.8 mV to 26.9 ± 2.9 mV (Delta Vrev = +26.8 mV). Both of these findings indicate that a significant component of the ion current passing through the 5-HT3A-STM receptor is carried by Cl- ions. These data therefore suggest that the cation-selective WT 5-HT3A receptor has been converted to an anion-selective receptor by the introduction of the three point mutations.



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Fig. 4.   Current-voltage relationships for WT and 5-HT3A-STM receptors. A and B, sample traces illustrating experiments used to determine the current-voltage relationships for the WT and 5-HT3A-STM receptors. 5-HT-evoked (10 µM, 2 s) current responses were recorded at the holding potentials shown. These traces are from experiments conducted in E2 (NaCl-mannitol; see "Materials and Methods") extracellular solution and are representative of five similar experiments. C, current-voltage relationships determined from the data like that shown in A and B for WT and 5-HT3A-STM receptors in E1 (normal extracellular), E2 (NaCl-mannitol) and E3 (NaCl-isethionate) solutions are shown. The resulting current-voltage relationships were corrected for liquid junction potentials post hoc according to the method of Barry and Lynch (20), and the mean reversal potentials determined from these data sets are given in Table II.


                              
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Table II
Reversal potential data for WT and 5-HT3A-STM receptors
Data shown are the mean ± S.E. (n = 4-5). Data have been corrected for liquid junction potentials according to the method of Barry and Lynch (20).

To accurately quantify this change in ion selectivity and allow calculation of the permeability ratio PCl/PNa for both WT and 5-HT3A-STM receptors, we performed a series of further experiments using simplified solutions in which Na+ was the predominant cation (see "Materials and Methods"), and other monovalent and divalent cations were eliminated or reduced as far as possible. In these experiments, the current-voltage relationships were more conveniently established by the use of voltage ramp protocols (-70 to +100 mV, in 200 ms) applied during the plateau phase of an agonist-induced response. The resulting data obtained for the WT receptor shows the expected inward rectification profile for the 5-HT3A receptor and a reversal potential close to 0 mV (-0.6 ± 0.3 mV) in symmetrical NaCl (Fig. 5). Substitution of 90% of the extracellular chloride with isethionate did not have any significant effect on the reversal potential of the WT receptor (Delta Vrev = 1.2 ± 1.2 mV), consistent with it being essentially impermeable to Cl- ions. In fact, a shift in the reversal potential (Delta Vrev) of less than 1 mV under such conditions is consistent with a PCl/PNa of <0.05 or a PNa/PCl of >20. Equivalent experiments on the 5-HT3A-STM receptor showed a reversal potential close to 0 mV in symmetrical NaCl, and this was shifted to 41.0 ± 2.5 mV by the substitution of of 90% of the extracellular chloride with isethionate. The shift in the reversal potential (Delta Vrev) of 43.7 ± 3.5 mV is consistent with a PCl/PNa of 12.3 or a PNa/PCl of 0.08 and convincingly demonstrates that the 5-HT3A-STM receptor is selective for chloride and relatively impermeable to sodium.



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Fig. 5.   Determination of the ion selectivity of the WT and 5-HT3A-STM receptors. Data obtained from voltage ramp protocols for WT and 5-HT3A-STM receptors in symmetrical NaCl solutions (A, solution E4; see "Materials and Methods") and upon substitution of 90% of the extracellular chloride by isethionate (B, solution E5). Voltage ramps (-70 to +100 mV in 200 ms) were applied during the plateau phase of an agonist-induced response (10 µM 5-HT) and under control conditions (no 5-HT) The current-voltage curves shown represent the net 5-HT-evoked current after any leak currents (control ramps) have been subtracted. Individual data sets were normalized to the current recorded at -70 mV and then averaged. Occasional error bars from the averaging process are shown. The resulting current-voltage relationships were corrected for liquid junction potentials post hoc according to the method of Barry and Lynch (20). The reversal potentials were determined individually for each current-voltage relationship before averaging, and the mean values obtained for each solution are shown below the relevant current-voltage curve. The shift in reversal potential as a result of the reduction in extracellular chloride Delta Vrev is also shown. This was not significant for the WT receptor, consistent with its known cation selectivity. In contrast, a large Delta Vrev was seen for 5-HT3A-STM, consistent with the construction of a chloride-selective receptor.



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ABSTRACT
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MATERIALS AND METHODS
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The results presented here show that the introduction of three point mutations in or near the M2 domain are sufficient to convert the ion selectivity of homomeric 5-HT3A receptors from cationic to anionic. Substitution of V13'T in M2 together with the neutralization of a ring of glutamate residues (E-1'A) and the adjacent insertion of the "extra" amino acid (proline) found in the anionic channels (referred to here as P-1") in the M1-M2 loop (Fig. 1) lead to the formation of an anion-selective 5-HT3A receptor. These mutations also had a significant effect on other properties of the 5-HT3A receptor, and these are discussed below.

Gating and Desensitization of the 5-HT3A-STM-- Ligand-gated ion channels, exemplified by the nACh receptor, open rapidly following agonist binding and, in the continued presence of agonist, may enter an agonist-bound but nonconducting "desensitized" state (23); this desensitized state of a receptor possesses a higher affinity for agonist than the closed resting or open states of the channel. Interestingly, 5-HT3A-STM receptors exhibited a higher apparent affinity for 5-HT compared with WT, and the whole-cell currents recorded exhibited a marked reduction in the rate of activation and desensitization. In fact, the EC50 for 5-HT on the 5-HT3A-STM receptor (240 nM) is similar to the value of 230 nM reported for the EC50 of the alpha 7-2 mutant nACh receptor for acetylcholine (1) and the estimated affinity of 5-HT for the desensitized state of the 5-HT3 receptor (50 nM) (27). The relevance of the lower Hill coefficient noted for the 5-HT3A-STM receptor (1.45), compared with WT is not clear but may reflect an alteration to the normal gating mechanism of the receptor. For instance, large variation in Hill coefficients (1.6-3.4) were noted for L9' substitutions of the 5-HT3 receptor reported by Yakel et al. (9), and Hill coefficients ranging between 1.4 and 2.8 were reported by Galzi et al. (1) for various channel lining mutants in the alpha 7 nACh receptor.

The effects of the 5-HT3A-STM receptor mutations on the time course of 5-HT-gated whole-cell currents are strikingly similar to the effects of the equivalent mutations on ACh-gated currents of alpha 7 nACh receptors (1). In this, and other studies (1, 24, 25), the combination of slower activation, reduced desensitization and increased affinity has led to the suggestion that the mutant receptor may attain a desensitized but conducting state (D*) in the presence of agonist. The slow decay of 5-HT3A-STM receptor whole-cell current responses could therefore be due to a similar phenomenon. If the mutations have changed one (or more) of the desensitized states of the receptor into a conducting one, then the reduced rate of current decay may reflect passage from the open to a conducting desensitized state before subsequent entry into a further (nonconducting) desensitized state. Such a model may also explain the long duration open times observed even in responses to short pulses of agonist (Fig. 3E), where the receptor may remain in a high affinity desensitized (conducting) state until agonist unbinding occurs. Further work, including single channel recording, has established unequivocally that the mutant nACh receptor can indeed attain a desensitized conducting state; it shows spontaneous channel activity and can even be gated by the competitive nACh receptor antagonist dihydro-beta -erythroidine (1, 26). Attempts to gather similar evidence for the existence of a desensitized conducting state of the 5-HT3A receptor were hampered by the low single channel conductance of the homomeric receptor. Although we have previously been able to use fluctuation noise analysis to study the unitary conductance of the WT and mutant 5-HT3 receptors (19), preliminary studies on the 5-HT3A-STM receptor failed to detect any resolvable channel noise (alternating current signal).2 These findings may therefore reflect a reduced single channel conductance of the receptor, but further studies would be required to support this conclusion. It might, however, be anticipated that the conductance of the mutant receptor would be low, since we have mutated one of the rings of charged residues that Imoto et al. (12) has shown affect conductance in the nACh receptor. In addition, it is likely that the inner and outer vestibules of the channel play a role in concentrating the appropriately charged ionic species (28); since we did not modify these regions, the 5-HT3A-STM receptor may inadvertently be designed to effectively concentrate ions of the wrong charge. Indeed, similar observations reported for the converse glycine receptor STM described by Keramidas et al. (2) make this an attractive hypothesis. We also investigated if D-tubocurarine or granisetron, both of which are antagonists of the 5-HT3 receptor, could activate 5HT3A-STM, but both compounds were inactive in this respect. Furthermore, the use of D-tubocurarine or granisetron did not provide any evidence for the occurrence of spontaneous channel activity, since they had no effect on the small resting leak currents observed in whole-cell recordings on the 5-HT3A-STM receptor. Therefore, whether or not the 5-HT3 and alpha 7 nACh receptors share an ability to attain a D* state is still not resolved and warrants further investigation. The recent identification of 5-HT3B, which can give rise to 5-HT3A/B heteromers with increased single channel conductance (~16 pS) compared with the 5-HT3A homomer (5) may prove useful in this respect. Furthermore, the likelihood of alpha 7 nACh receptor mutants exhibiting spontaneous activity appears to be linked to the V13' substitution (26); alternative mutations to V13'T within the 5-HT3 receptor may therefore be an interesting starting point for further work and may provide evidence for subtly different gating mechanisms between the two receptors. In fact, a recent report describing a 5-HT3A receptor V13'S mutation supports this idea, since this receptor has a similar EC50 and slowly desensitizing phenotype to the STM described here and yet also exhibits clear spontaneous gating behavior (29).

Ion Selectivity of the 5-HT3A-STM-- Current-voltage relationships for the WT and 5-HT3A-STM receptors exhibited similar, inward, rectification in all extracellular solutions used. The reversal potential for the WT receptor was shifted to a more negative potential by the reduction in extracellular NaCl concentration (Fig. 4) and was not significantly altered when extracellular Cl- was largely substituted with isethionate. This, in agreement with other findings (30-35), indicates that the current passing through the WT receptor is predominantly carried by Na+ ions. In contrast, equivalent experiments on the 5-HT3A-STM receptors showed reversal potential shifts to positive potentials, consistent with an appreciable chloride permeability of the receptor. Use of simplified, symmetrical NaCl conditions to allow accurate determination of the chloride permeability of the WT and 5-HT3A-STM receptors further supported these conclusions, showing that the insignificant chloride permeability of the cation-selective WT receptor (PCl/PNa < 0.05) was replaced by a highly chloride permeable channel in the 5-HT3A-STM receptor (PCl/PNa = 12.3; PNa/PCl = 0.08). The three mutations introduced into the WT receptor are therefore sufficient to switch the selectivity of the 5-HT3 receptor from cationic to anionic. These results complement those from the alpha 7 nACh and glycine receptor studies (1, 2) in which the same three mutations were capable of producing similarly dramatic changes in ion selectivity. For instance, in the case of the glycine receptor study the chloride permeable WT receptor (PNa/PCl = 0.04; PCl/PNa = 24.6) was converted to a sodium-selective channel with a PNa/PCl of 3.7 (PCl/PNa = 0.27) by the "reverse" set of mutations. The properties of the three amino acids that make up the STM and their location in or near M2 therefore have important implications for the understanding of channel gating and ion selectivity of the entire LGIC superfamily.

Structural Implications and Location of the Charge Selectivity Filter-- Perhaps the most striking feature of the STM is the fact that two of the three amino acids are not technically within the M2 domain at all (as defined in Fig. 1) but instead reside in the M1-M2 loop. This not only highlights the importance of this short linker segment in normal receptor function but also strongly suggests that this region of the protein may contribute directly to the ion conduction pathway. The greatest insight into the individual roles of the amino acids that make up the STM has been provided by further work reported by Corringer et al. (26) on the charge selectivity filter of the alpha 7 nACh receptor, which demonstrates an essential role of the inserted proline residue in the resultant anionic phenotype. In their model, the E-1'A and V13'T mutations are considered to be "permissive" rather than "essential" for the conversion in ion selectivity; the E-1'A/V13'T double mutant does not change the ion selectivity, but these changes combined with the addition of the proline result in the receptor becoming anion-selective (26). Thus, the critical difference between the cationic and anionic channels is the introduction of this proline residue in the M1-M2 intracellular loop and is consistent with the idea that a large structural rearrangement is likely to underlie the dramatic change in ion selectivity of the channel rather than the addition or removal of charged amino acid side chains. What is not clear at present is over what distance such a structural rearrangement may occur. Is it just at the level of the intracellular mouth of the channel, or does it extend to a rearrangement of part or all of the M2 domain as well? Further mutagenesis experiments have been designed to address this point and do show that the inserted proline residue can generate functional anionic receptors even when inserted at the -3' or -1' positions (26); however, insertion at -4' resulted in cationic receptors, and insertion at 0' resulted in nonfunctional receptors. Clearly, a higher resolution structure of the channel region of the protein is now almost a prerequisite for an interpretation of the effects of such mutations.

The importance of the arrangement of the polypeptide backbone, rather than the properties of individual amino acid side chains, in the determination of the ion selectivity has now been demonstrated for the voltage-gated K+ channel (36). Based on the structure of this channel, solved to 3.2-Å resolution, Doyle et al. (36) suggested that the K+ selectivity results from the specific coordination of K+ ions by a ring of main chain carbonyl atoms positioned at the selectivity filter of the channel. Similar structural features may be important in the selectivity filter of LGICs. So far, the structure of the ACh receptor, originally solved to 9 Å by electron microscopy (37) has now been refined to 4.6 Å (38), and future work may yet provide sufficient improvements in resolution to pinpoint regions of secondary structure within the channel mouth and gate. Such studies have already allowed insight into the structural reorganization that may occur upon channel gating (39) and may yet allow insight into the further changes that no doubt occur (40) upon receptor desensitization.

In conclusion, the results of this study, combined with the findings of Yakel et al. (9) and Eiselé et al. (8), which showed that the gating mechanisms of the 5-HT3 and alpha 7 nACh receptors were likely to be conserved, highlight the striking level of conservation of the molecular mechanisms underlying the functioning of these and related members of the LGIC superfamily. Additionally, now that an equivalent study has been reported for the glycine receptor (2), it is clear that the determinants of cation versus anion selectivity identified here represent a conserved feature of the LGIC superfamily.


    ACKNOWLEDGEMENTS

We thank Andrew Randall, Elizabeth Fletcher, John Peters, and Nigel Unwin for advice or comment on various aspects of the study and Graham Smith for technical assistance.


    FOOTNOTES

* A preliminary report of these findings has been published in abstract form (15).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Recipient of a Medical Research Council studentship. Present address: Neuroscience Dept., SmithKline Beecham Pharmaceuticals, New Frontiers Science Park (North), Harlow, Essex CM19 5AW, United Kingdom.

|| A Wellcome Trust Senior Research Fellow in Basic Biomedical Science. To whom correspondence should be addressed: Dept. of Biochemistry, Tennis Court Rd., Cambridge CB2 1QT, United Kingdom. Tel.: 44 1223 766047; Fax: 44 1223 402310; E-mail: s.lummis@mole.bio. cam.ac.uk.

Published, JBC Papers in Press, January 3, 2001, DOI 10.1074/jbc.M009575200

2 M. J. Gunthorpe and J. A. Peters, unpublished observations.


    ABBREVIATIONS

The abbreviations used are: 5-HT3, 5-hydroxytryptamine3; LGIC, ligand-gated ion channel; nACh, nicotinic acetylcholine; STM, selectivity triple mutation; WT, wild type.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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