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J. Biol. Chem., Vol. 283, Issue 28, 19301-19313, July 11, 2008

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Structural Determinants of Ca²⁺ Permeability and Conduction in the Human 5-Hydroxytryptamine Type 3A Receptor^*

Matthew R. Livesey¹, Michelle A. Cooper, Tarek Z. Deeb, Jane E. Carland, Janna Kozuska, Tim. G. Hales^¶, Jeremy J. Lambert², and John A. Peters²³

From the Neurosciences Institute, Division of Pathology and Neuroscience, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom and the Departments of Pharmacology and Physiology and ^¶Anesthesiology and Critical Care Medicine, George Washington University, Washington, D. C. 20037

Received for publication, March 27, 2008 , and in revised form, May 8, 2008.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cation-selective cysteine (Cys)-loop transmitter-gated ion channels provide an important pathway for Ca²⁺ entry into neurones. We examined the influence on Ca²⁺ permeation of amino acids located at intra- and extracellular ends of the conduction pathway of the human 5-hydroxytryptamine type 3A (5-HT_3A) receptor. Mutation of cytoplasmic arginine residues 432, 436, and 440 to glutamine, aspartate, and alanine (the aligned residues of the human 5-HT_3B subunit (yielding 5-HT_3A(QDA)) increased P_Ca/P_Cs from 1.4 to 3.7. The effect was attributable to the removal of an electrostatic influence of the Arg-436 residue. Despite its relatively high permeability to Ca²⁺, the single channel conductance of the 5-HT_3A(QDA) receptor was depressed in a concentration-dependent and voltage-independent manner by extracellular Ca²⁺. A conserved aspartate, located toward the extracellular end of the conduction pathway and known to influence ionic selectivity, contributed to the inhibitory effect of Ca²⁺ on macroscopic currents mediated by 5-HT_3A receptors. We introduced a D293A mutation into the 5-HT_3A(QDA) receptor (yielding the 5-HT_3A(QDA D293A) construct) to determine whether the aspartate is required for the suppression of single channel conductance by Ca²⁺. The D293A mutation decreased the P_Ca/P_Cs ratio to 0.25 and reduced inwardly directed single channel conductance from 41 to 30 pS but did not prevent suppression of single channel conductance by Ca²⁺. The D293A mutation also reduced P_Ca/P_Cs when engineered into the wild-type 5-HT_3A receptor. The data helped to identify key residues in the cytoplasmic domain (Arg-436) and extracellular vestibule (Asp-293) that markedly influence P_Ca/P_Cs and additionally directly demonstrated a depression of single channel conductance by Ca²⁺.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The 5-hydroxytryptamine type 3 (5-HT₃)⁴ receptor is a cation-selective ligand-gated ion channel of the Cys-loop superfamily with structural and functional homology to the family of nicotinic acetylcholine (ACh) receptors (1–3). 5-HT₃ receptor activation elicits rapidly activating and desensitizing inward current responses mediated by the flux of both mono- and divalent cations (2). 5-HT₃ receptor stimulation contributes to fast excitatory synaptic transmission in the central nervous system (4–7) and also modulates the release of several neurotransmitters including acetylcholine, cholecystokinin, dopamine, glutamate, norepinephrine, and particularly -aminobutyric acid, the exocytosis of which is enhanced by direct Ca²⁺ influx through the ionophore of presynaptic 5-HT₃ receptors (8, 9).

5-HT₃ receptors can be assembled in vitro as pentamers from a selection of five gene products, namely the 5-HT_3A, 5-HT_3B, 5-HT_3C, 5-HT_3D, and 5-HT_3E subunits (10–16). The 5-HT_3A subunit assembles as a functional homopentamer and is mandatory for cell surface expression of any of the other 5-HT₃ receptor subunits (10, 14, 17). Only homopentameric 5-HT_3A and heteropentameric 5-HT_3A/5-HT_3B subunit complexes have been characterized in detail (10–12). These two receptor types differ in their sensitivity to activation by 5-HT, antagonism by certain agents, and allosteric modulation by some general anesthetics and 5-substituted indole analogues (11, 12, 18, 19). Strikingly, the human homomeric 5-HT_3A receptor exhibits an unusually small single channel conductance within the range of 0.3 to 1 pS (20–22), whereas the corresponding range of 16 to 30 pS for the human 5-HT_3A/5-HT_3B heteromer (11, 23) is more akin to the single channel conductance of most native 5-HT₃ receptors and other members of the Cys-loop receptor superfamily family. Heteromeric 5-HT_3A/5-HT_3B receptors additionally differ from the homomeric 5-HT_3A isoform by exhibiting a reduced relative permeability to Ca²⁺, no measurable permeability to Mg²⁺, faster agonist-induced desensitization kinetics, and a propensity to spontaneous channel opening (11, 12, 18). Diversity within the human 5-HT_3A and 5-HT_3B subunit structure occurs through alternative splicing and polymorphisms, some of which impact significantly upon receptor expression and function and are linked to human pathologies (23–25).

Cys-loop receptor subunits share a common overall topology of extracellular, transmembrane (TM), and intracellular domains, which represent functionally interacting modules (26, 27) (Fig. 1A). The TM domain consists of four -helices (TM1–4), the second of which (TM2) lines the ion channel (26, 27). A wealth of evidence across both cation- and anion-selective channels of the Cys-loop family implicate specific amino acid residues within TM2 and the adjacent sequences as fundamental determinants of ionic selectivity and single channel conductance (2, 26, 28–30) (Fig. 1B).

Notwithstanding the importance of the TM2 domain, we have identified an additional -helical structure (the MA helix) within the large intracellular loop linking TM3 and TM4 that greatly influences single channel conductance (2, 22, 31, 32) (Fig. 1C). In particular, the peculiarly small single channel conductance of the human homomeric 5-HT_3A receptor is due to the unique presence of three arginine residues (Arg-432, Arg-436, and Arg-440) that impede cation conductance by both steric and repulsive electrostatic influences (32). The combined substitution of these residues by those aligned in the human 5-HT_3B receptor subunit (i.e. R432Q, R436D, R440A, generating a construct coined 5-HT_3A(QDA)) enhances single channel conductance by 40-fold (22, 33) (Fig. 1C). Importantly, arginine residues engineered into the corresponding locations of both subunit species of the nicotinic ACh ₄β₂ receptor depress single channel conductance (22). Such findings have been rationalized in the light of the 4 Å resolution model of the nicotinic ACh receptor of Torpedo marmorata in which the five MA-stretch helices manifest as an inverted pentagonal cone that projects from the plasma membrane into the cytoplasm forming the intracellular vestibule of the channel (26). Narrow (8 Å) lateral windows (or "portals") situated at the subunit interfaces, which are lined by the MA helices, form an obligate pathway for ion permeation (26) (Fig. 1A).

Here, we demonstrate that residues within the MA helices also strongly affect the permeability of Ca²⁺ relative to monovalent ions (e.g. P_Ca/P_Cs) but do not influence charge selectivity (e.g. P_Na/P_Cl). Additionally, we describe the influence of Ca²⁺ on 5-HT-evoked single channel conductance using the 5-HT_3A(QDA) construct as a model, which, unlike the wild-type receptor, mediates unitary events that can be directly resolved in outside-out patch recordings. Finally, we have examined the role of an aspartate residue, located at the extracellular end of the conduction pathway, which is known to influence ionic conductance and selectivity in other Cys-loop receptors (28) and contributes to an inhibitory effect of Ca²⁺ on macroscopic currents mediated by 5-HT_3A receptors (34). The introduction of a D293A mutation into the 5-HT_3A(QDA) receptor (yielding the 5-HT_3A(QDA D293A) construct) markedly suppressed P_Ca/P_Cs and reduced inwardly directed single channel conductance.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
5-HT_3A Receptor Constructs and Transfection of cDNAs—cDNAs encoding human wild-type and mutant 5-HT_3A receptor subunits were cloned into pGW1 (17). Point mutations were introduced using standard molecular biological techniques (31), and all cDNAs were sequenced to confirm fidelity. Wild-type and mutant receptor subunit cDNAs were co-transfected into tsA-201 or HEK-293 cells, with a cDNA encoding green fluorescent protein to identify transfected cells. Transfection was performed by electroporation (400 V, 125 microfarads, infinite resistance) using a Bio-Rad Gene Electropulser II (BioRad) or the calcium phosphate precipitation method. Transfected cells were routinely cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 100 µg ml^–1 streptomycin, and 100 units ml^–1 penicillin and maintained at 37 °C for 24–48 h in 95% air, 5% CO₂ at 100% humidity before use.

Electrophysiological Recordings—Whole-cell and outside-out patch configurations were used to record macroscopic and single channel currents, respectively, from transfected cells. All experiments were performed at room temperature (20–23 °C). The recording chamber was routinely superfused (5 ml min^–1) with an extracellular solution (E1) comprising (in mM): NaCl 140, KCl 2.8, MgCl₂ 2.0, CaCl₂ 1.0, glucose 10, HEPES 10 (pH 7.2, adjusted with 1 M NaOH; final [Na⁺]_o = 146 mM). Patch electrodes (resistance = 2–8 megohms when measured in solution E1) were filled with an intracellular solution (I1) containing (in mM): CsCl 140, CaCl₂ 0.1, EGTA 1.1, HEPES 10 (pH 7.2, adjusted by 1 M CsOH, final [Cs⁺]_i = 143 mM). In solution I2, CsCl was totally replaced by NaCl (pH 7.2, adjusted by 1 M NaOH, final [Na⁺]_i = 145 mM). The intracellular free calcium concentration ([Ca²⁺]_i) for I1 and I2 was estimated to be 10 nM (35). To determine the relative permeability of Na⁺ versus Cs⁺ (P_Na/P_Cs), in solution E2, additional NaCl totally replaced KCl in the extracellular solution, and the concentrations of CaCl₂ and MgCl₂ were each reduced to 0.1 mM to minimize the influence of divalent cations upon the reversal potential of the macroscopic current response to 5-HT (E_5-HT). The permeability of Ca²⁺ relative to Cs⁺ (P_Ca/P_Cs) was determined using an extracellular solution (E3) containing (in mM): CaCl₂ 100, L-histidine 5, glucose 10 (pH 7.2) (20). To prevent changes in reference electrode potential during the superfusion of media with altered ionic composition, a bridge containing 3 M KCl in agar (4% w/v) was employed. Liquid junction potentials arising at the tip of the patch pipette were measured as described by Fenwick et al. (35), and potential measurements were corrected post hoc.

E_5-HT was determined by two methods. In the first, the membrane potential was stepped from –60 mV to –100 mV for 100 ms and subsequently ramped to +60 mV within 1 s at the peak of the macroscopic current response to pressure-applied 5-HT (1 µM). Care was taken to ensure that the agonist-induced current recorded at –60 mV before and immediately following the voltage ramp did not change. Subtraction of the leakage current recorded in the absence of 5-HT from the current recorded in the presence of the agonist yielded the current-voltage (I-V) relationship attributable to the 5-HT-evoked conductance increase. In the second method, peak current responses to pressure-applied 5-HT (10 µM) were recorded at steady holding potentials closely bracketing E_5-HT, which was subsequently determined by interpolation. The two methods gave similar results.

Concentration-response relationships to 5-HT (0.3–100 µM) were determined from the peak inward whole-cell current response recorded in the presence of solutions E1 and I1 at a holding potential of –60 mV. The data were fitted iteratively using SigmaPlot 8 (Systat Software Inc., San Jose, CA) by an equation of the form

(Eq. 1)

where I is the peak current elicited by 5-HT at concentration [A], I_max is the peak current evoked by a saturating concentration of 5-HT, EC₅₀ is the concentration of 5-HT that evokes a half-maximal response, and n_H is the Hill coefficient. To combine the concentration-response data obtained from several cells, agonist-evoked currents were normalized and expressed as a percentage of the maximal inward current response produced by a saturating concentration of 5-HT obtained for each cell.

Single channel currents evoked by pressure application of 5-HT (10 µM) were recorded from excised outside-out membrane patches clamped at steady holding potentials within the range of –100 to +100 mV as specified under "Results." The solutions used in such experiments were I1 and E1, E2, and E3, as appropriate. Additionally, in experiments that determined E_5-HT and single channel conductance in mixtures of extracellular Na⁺ and Ca²⁺, NaCl was held at a constant concentration of 95 mM in the presence of variable concentrations of CaCl₂ (10^–8–3 x 10^–2 M), sucrose (0–90 mM, as appropriate to maintain constant osmolarity), glucose (10 mM), and L-histidine (5 mM; pH 7.2). Currents were recorded using an Axopatch-1D amplifier (Axon Instruments, Union City, CA), low pass-filtered (5 KHz, Bessel characteristic), and recorded onto magnetic tape using a Bio-Logic DAT recorder (Bio-Logic, Claix, France) for subsequent offline analysis.

Data Analysis—Permeability ratios (relative to Cs⁺) were determined from measurements of E_5-HT and calculated ion activities (i.e. the ionic concentration multiplied by ion activity coefficient (_ion)). The latter were estimated (following the Guggenheim convention) to be: _Cs 0.72 (140 mM Cs⁺) _Na 0.76 (140 mM Na⁺), and _Ca 0.26 (100 mM Ca²⁺). For varying concentrations of Ca²⁺ in the presence of 95 mM Na⁺, the mean molal activity coefficients for CaCl₂ in the presence of 100 mM NaCl, as tabulated by Butler (36), allowed calculation of _Ca. Sucrose had a negligible influence upon _Na over the range of sugar concentrations employed (37). The permeability ratio P_Na/P_Cs was calculated from the Goldman-Hodgkin-Katz (voltage) equation,

(Eq. 2)

where R, T, and F have their usual meaning and [Na⁺]_o and [Cs⁺]_i are the calculated activities of extracellular Na⁺ and internal Cs⁺ ions, respectively. This equation ignores the very small error anticipated due to the presence of low concentrations of permeant divalent ions (e.g. Ca²⁺) within the extra- or intracellular solutions.

The permeability ratio, P_Ca/P_Cs, was calculated from a modified Goldman-Hodgkin-Katz (voltage) equation (38) which, with both Na⁺ and Ca²⁺ present as permeant species in the extracellular medium but only Cs⁺ and negligible Ca²⁺ present in the pipette (`intracellular') solution, can be written as (20)

(Eq. 3)

where [Na⁺]_o and [Ca²⁺]_o are the external activities of Na⁺ and Ca²⁺, [Cs⁺]_i is the internal activity of Cs⁺, and P'_Ca/P_Cs is a modified term relating the permeability of Ca²⁺ to Cs⁺. Substituting for P'_Ca/P_Cs, the above can also be written as

(Eq. 4)

When Ca²⁺ was the sole cation in the extracellular medium, the term P_Na/P_Cs was omitted. For simplicity, in the text we routinely refer to ion concentrations (in square brackets) rather than activities (in parentheses), although the latter were used in all calculations of relative permeability and are presented in the figures where relevant.

Single channel currents were low pass-filtered offline at 1 KHz, digitized at 10 KHz via a DigiData 1302A (Axon Instruments) interface. Using the WinEDR V2 7.6 electrophysiology data recorder (J. Dempster, Dept. of Physiology and Pharmacology, University of Strathclyde, UK), single channel current amplitude histograms were constructed from sections of single channel activity in which unitary events predominated. A transition detection threshold of 30–40% of the predominant unitary event amplitude was employed to capture events for inclusion within the amplitude histogram. Events less than 1 ms in duration (classified as incompletely resolved), obvious artifacts, and those corresponding to multiple openings were rejected. The resulting mean open state amplitude histogram was fitted using an iterative least squares algorithm with a single Gaussian probability density function from which unitary event amplitude (i) was determined. The analysis was restricted to the dominant unitary current state, as events of differing amplitude were relatively rare. Single channel conductance () is routinely reported as the chord conductance, i.e. = i/(V_m – E_5-HT), where V_m is the holding potential (including liquid junction potential correction) and E_5-HT is the reversal potential of the agonist-evoked macroscopic response determined as described above under the appropriate ionic conditions.

Statistical Analysis—Data are presented as mean ± S.E. Statistical analysis was conducted using one-way analysis of variance with the post hoc Dunnett or Tukey test as appropriate. A value of p < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Residues in the MA helix of the 5-HT_3A Receptor Influence Ca²⁺ Permeability—We recently demonstrated that arginine residues 432, 436, and 440 within the MA helix motif of the TM3–4 loop at positions termed MA –4', 0', and 4', respectively, which line putative cytoplasmic portals (Fig. 1C), are responsible for the sub-pS single channel conductance of the 5-HT_3A receptor (2, 22, 31). We began this study by testing the hypothesis that the same 5-HT_3A MA helix arginine residues also serve as determinants of ionic selectivity.

View larger version (17K): [in this window] [in a new window]   FIGURE 1. Domains of the 5-HT3 receptor that influence ion conduction and selectivity between mono- and divalent cations. A, homology model of the wild-type 5-HT3A receptor using the nicotinic ACh receptor of Torpedo marmorata as a template (32). The functional modules are denoted: extracellular domain (EC), poreforming transmembrane helices (TM), and the portal-framing cytoplasmic MA helices (MA). B, sequence alignment of the second transmembrane domain (TM2) and flanking sequences of the human 5-HT3A and 5-HT3B subunits. The residue mutated to alanine in the mutant 5-HT3A receptor constructs is boxed. C, an alignment of amino acids within the MA helices of wild-type 5-HT3A and 5-HT3B subunits and the mutant 5-HT3A(QDA) construct used in this study, indicating the positions of the –4', 0', and 4' residues (boxed). Amino acids are numbered according to the human 5-HT3A subunit amino acid sequence (51).

In subsequent experiments, we evaluated the contributions of the individual mutations R432Q (MA –4'), R436D (MA 0'), and R440A (MA 4') to the enhancement of P_Ca/ P_Cs. The R436D charge reversal mutation produces an 6-fold increase in single channel conductance compared with the wild-type 5-HT_3A receptor (22). The 5-HT_3A(R436D) receptor also had a significantly enhanced relative permeability to Ca²⁺ (P_Ca/P_Cs = 3.13 ± 0.31, n = 7, Fig. 3A, Table 1). By contrast, the introduction of aspartate did not significantly affect P_Na/P_Cs compared with wild-type 5-HT_3A receptors (Fig. 3A, Table 1). Notably, the P_Ca/P_Cs of the 5-HT_3A(QDA) receptor was not significantly greater than that of the 5-HT_3A(R436D) receptor. The individual replacement of Arg-432 by the equivalent 5-HT_3B subunit residue glutamine had no significant effect on single channel conductance (22). Likewise, the permeability of Ca²⁺ relative to Cs⁺ of the 5-HT_3A(R432Q) mutant was similar to that of the wild-type 5-HT_3A receptor, suggesting that this MA helix residue has little influence on ion selectivity (Fig. 3A, Table 1). Substitution of Arg-440 residue by alanine significantly increased single channel conductance (22). Although the 5-HT_3A(R440A) mutant exhibited a trend toward an increased P_Ca/P_Cs ratio relative to the wild-type receptor, the effect failed to reach statistical significance (Fig. 3A, Table 1).

The validity of the above estimates of P_Na/P_Cs and P_Ca/P_Cs for the 5-HT_3A(QDA) receptor construct relies upon the mutant retaining the near perfect cation versus anion selectivity of the wild-type receptor (P_Na/P_Cl = 53) (30). We tested this assumption rigorously by dilution experiments in which the NaCl content of the extracellular solution E2 was reduced to 95, 50, and 20 mM by replacement with sucrose. Under such conditions a plot of E_5-HT as a function of the logarithm of extracellular Na⁺ activity, (Na⁺)_o, was linear and yielded a slope of 59 mV/decade change in (Na⁺)_o (Fig. 3B). A similar dependence upon (Na⁺)_o was observed under simplified bi-ionic conditions in which Na⁺ totally replaced Cs⁺ within the intracellular solution. Because only a slope of less than 58 mV/decade change in (Na⁺)_o is compatible with anion permeation (28), we concluded that for the 5-HT_3A(QDA) receptor construct P_Na/P_Cl = .

The foregoing results indicate that the arginine residues present at positions 436 (MA 0') and to a lesser extent 440 (MA 4') of the human wild-type 5-HT_3A receptor collectively suppress the permeability of Ca²⁺, but not Na⁺, relative to Cs⁺, revealing that the large intracellular loop of a Cys-loop receptor is an important determinant of divalent versus monovalent cation selectivity.

View larger version (16K): [in this window] [in a new window]   FIGURE 2. The reversal potential of 5-HT-evoked macroscopic currents at wild-type 5-HT3A and 5-HT3A(QDA) receptor constructs with Na+ or Ca2+ as the charge carrier in the extracellular solution. A, voltage ramp (left) applied at the peak of the macroscopic inward current response to pressure-applied 5-HT (1 µM)(right) to determine the reversal potential (E5-HT) of the agonist-induced response mediated by the wild-type 5-HT3A receptor. B, current-voltage relationships recorded from a representative HEK-293 cell expressing the wild-type 5-HT3A receptor with an intracellular CsCl-based solution (I1) as reference and NaCl (146 mM)-based solution E2 or CaCl2 (100 mM)-based solution E3 as the extracellular electrolyte. Note the negative shift in E5-HT (arrow) upon complete iso-osmotic replacement of extracellular Na+ by Ca2+. C, current-voltage relationships recorded from a representative HEK-293 cell expressing the 5-HT3A(QDA) receptor under the conditions described in B. Note that complete iso-osmotic replacement of extracellular Na+ by Ca2+ causes a positive shift in E5-HT (arrow).

View larger version (10K): [in this window] [in a new window]   FIGURE 3. Comparison of the relative ion permeability of wild-type and mutant 5-HT3A receptor constructs. A, bar graph illustrating the relative permeability (Px/PCs) of Na+ (black bars), or Ca2+ (white bars) relative to Cs+ for the indicated receptor constructs. Permeability ratios were determined with I1 as the intracellular reference solution and NaCl (146 mM)-based solution E2 or CaCl2 (100 mM)-based solution E3 as the extracellular electrolyte. Data are the mean ± S.E. of 5–17 independent estimates of Px/PCs performed from separate cells. Mutant constructs in which PNa/PCs or PCa/PCs was significantly different from the appropriate wild-type control are indicated with an asterisk (p < 0.05; analysis of variance followed by Dunnett's post hoc test). B, relationship between the reversal potential of the macroscopic current evoked by 5-HT (E5-HT) and the activity of Na+ in the extracellular medium ((Na+)o) for the 5-HT3A(QDA) receptor construct. Responses were recorded with extracellular electrolytes based on solution E2 wherein the concentration of NaCl was reduced by substitution with sucrose. Intracellular solutions were either CsCl-based I1 (filled circles) or a solution in which NaCl totally replaced CsCl (open circles) to produce bi-ionic recording conditions. The regression line fitted to the filled circles yielded a slope of 59 mV/decade change in (Na+)o. Data points are the mean of 3–6 independent determinations of E5-HT, and vertical bars indicate ± S.E. C, relationship between PNa/PCs (filled circles) and PCa/PCs (open circles) and the charge of the amino acid residue at position 436 of the MA helix.

With Na⁺ as the predominant extracellular cation, the single channel current-voltage relationship (i-V) for the 5-HT_3A (QDA) receptor shows a very mild outward rectification over a wide range of holding potentials (–100 to 100 mV) and yields chord conductance levels of 46 and 40 pS at holding potentials of +100 and –100 mV, respectively (Fig. 4, A and B). The latter is slightly larger than previously reported by us (22); this minor difference is due to the reduced concentration of divalent cations in the extracellular medium E2. Inclusion of 1 mM Ca²⁺ and 2 mM Mg²⁺ (medium E1) also yielded an essentially linear i-V relationship but with a slope conductance corresponding to our previous estimate (22).

In contrast to the mild outward rectification of the i-V relationship observed with Na⁺, when Ca²⁺ was the sole extracellular cation, 5-HT-evoked single channel events mediated by the 5-HT_3A(QDA) receptor displayed marked outward rectification (Fig. 4, A and B) similar to that seen in whole-cell current recordings (Fig. 2C). At all negative holding potentials, single channel current amplitudes were greatly reduced in comparison to when Na⁺ was the permeant cation. At the most negative potential studied (–100 mV), where it can be assumed that single channel currents reflect inward movement of Ca²⁺ with little opposing outwardly directed monovalent cation flux, a single channel chord conductance of 5.7 pS was calculated, assuming an E_5-HT of 9.4 mV obtained from macroscopic currents recorded under identical ionic conditions. The disparity between single channel current amplitudes recorded in Na⁺- and Ca²⁺-containing solutions decreased as the holding potential was progressively shifted to more positive values (Fig. 4, A and B). This is most clearly appreciated by inspection of Fig. 4C, which plots unitary current amplitude against driving force (i.e. holding potential minus E_5-HT). Indeed, at the most positive potentials examined, where an outwardly directed flux of Cs⁺ predominates, single channel current amplitudes were indistinguishable between Na⁺- and Ca²⁺ - based extracellular solutions (Fig. 4C). The data indicate that the 5-HT_3A(QDA) receptor construct, despite selecting for Ca²⁺ over Na⁺, conducts the former less efficiently, thus predicting that extracellular Ca²⁺ will (at negative holding potentials) reduce single channel conductance in mixtures of Na⁺ and Ca²⁺ as we previously inferred for the human wild-type 5-HT_3A receptor by fluctuation analysis (20).

View larger version (16K): [in this window] [in a new window]   FIGURE 4. Single channel currents mediated by the 5-HT3A(QDA) receptor construct with Na+ or Ca2+ as the extracellular permeant ion species. Single channel currents (i) were recorded from outside-out membrane patches in response to pressure-applied 5-HT (10 µM) at holding potentials (Vh) ranging from –100 to +100 mV. The patch pipette contained CsCl-based solution I1, and the patch was superfused with NaCl (146 mM)-based solution E2 or CaCl2 (100 mM)-based solution E3. A, exemplar single channel currents with either Na+ (left) or Ca2+ (right) as the extracellular permeant cation. B, current-voltage (i-V) plot for single channel currents recorded with NaCl-based solution E2 (filled circles) or CaCl2-based solution E3 (open circles) as the extracellular electrolyte. Note the mild outward rectification with Na+ as the permeant ion species in the extracellular medium (compare dashed line fitted to data obtained at positive potentials to curve fitted to all data points). By contrast, marked outward rectification is evident when CaCl2 totally replaces NaCl. C, i-V plot depicted in B but with driving force (i.e. Vh – E5-HT) replacing Vh on the abscissa. E5-HT was determined from macroscopic current responses to 5-HT (see"Experimental Procedures"). The plot reveals that outwardly directed single channel current amplitudes, at the most positive Vh values examined, are essentially identical in Na+- and Ca2+-containing extracellular solutions when equivalent driving forces are considered. Each data point in B and C is the mean of 3–10 determinations of single channel current amplitude derived from different outside-out membrane patches. The vertical bars, when exceeding the size of the symbols, indicate ± S.E.

Extracellular Ca²⁺ Reduces Single Channel Conductance of the 5-HT_3A(QDA) Receptor in a Voltage-independent Manner—Blockade by Ca²⁺ of macroscopic current responses mediated by the 5-HT_3A receptor expressed in mammalian cell hosts is voltage-independent (20, 21, 34, 40, 41). The readily resolvable single channel conductance of the 5-HT_3A(QDA) receptor allowed direct measurements of the influence of extracellular Ca²⁺ upon single channel current amplitudes over a range of membrane potentials. Fig. 6 depicts the results of such studies where [Ca²⁺]_o was varied between 0.3 and 10 mM in the presence of 95 mM Na⁺ and single channel events evoked by pressure-applied 5-HT (10 µM) were recorded from outside-out membrane patches at holding potentials in the range of –40 to –100 mV. At all concentrations of extracellular Ca²⁺ studied the single channel i-V relationship was well fitted by a linear function and showed no evidence of a region of negative slope conductance or outward rectification (Fig. 6). In addition, the single channel conductances derived from the slope of the i-V relationships (i.e. 35.8, 29.4, 25.0, and 20.0 pS in the presence of 0.3, 1, 3, and 10 mM Ca²⁺, respectively) were in excellent agreement with the chord conductances reported in Fig. 5C. Thus, the reduction in single channel conductance by extracellular Ca²⁺ is voltage-independent over the range of negative potentials examined.

View larger version (19K): [in this window] [in a new window]   FIGURE 5. Extracellular Ca2+ depresses the single channel conductance of the 5-HT3A(QDA) receptor construct. A, reversal potential of the macroscopic current response to 5-HT (E5-HT) with [Na+]o set to 95 mM and [Ca2+]o within the range of 0.1 to 30 mM. Note that Ca2+ ion activity, (Ca2+)o, rather than concentration, [Ca2+]o, is plotted in A and C. Data are the mean of 3–4 observations, and vertical bars indicate S.E. **, p < 0.01 as determined by analysis of variance followed by Dunnett's post hoc test. B, single channel currents in response to 5-HT (10 µM) applied by pressure to outside-out membrane patches voltage-clamped at a holding potential (Vh) of –80 mV. Increasing [Ca2+]o depresses unitary current amplitude in a concentration-dependent manner. Note that an increased driving force (i.e. Vh – E5-HT) when [Ca2+]o is equal to 10 and 30 mM (corresponding to a (Ca2+)o of 3.12 and 8.56 mM, respectively; see A) partially masks the magnitude of the suppression of single channel current amplitudes by Ca2+. C, concentration-response relationship illustrating the depressant effect of extracellular Ca2+ upon single channel conductance. Data are the mean ± S.E. of measurements performed on 4–5 outside-out membrane patches under the conditions described in B.

View larger version (26K): [in this window] [in a new window]   FIGURE 6. Extracellular Ca2+ reduces the single channel conductance of the 5-HT3A(QDA) receptor construct in a manner independent of transmembrane potential. A, single channel currents recorded from outside-out membrane patches in response to pressure-applied 5-HT (10 µM). The currents were recorded at holding potentials in the range of –100 to –40 mV in the presence of 0.3, 1, 3, or 10 mM extracellular Ca2+ and 95 mM Na+. B, i-V relationships for single channel events recorded with [Na+]o constant at 95 mM and [Ca2+]o present at 0.3 mM (filled circles), 1 mM (open circles), 3 mM (filled inverted triangles), or 10 mM (open inverted triangles). The relationship between single channel current amplitude and Vh at each concentration of Ca2+ was fitted by linear regression analysis to yield slope conductance values of 35.8, 29.4, 25.0, and 20.0 pS in the presence of 0.3, 1, 3, and 10 mM Ca2+, respectively. Dashed lines represent an extrapolation of each i-V plot to the zero current level (E5-HT). Each data point is the mean of single channel current amplitudes derived from recordings performed on 3–5 outside-out membrane patches, and the vertical bars indicate ± S.E.

View larger version (16K): [in this window] [in a new window]   FIGURE 7. The extracellular concentration of Na+ influences depression of single channel conductance by extracellular divalent cations. Bar graph depicting the single channel conductance of the 5-HT3A(QDA) receptor construct in the presence of 0.1 mM Ca2+ and 0.1 Mg2+ (black bars) or 1 mM Ca2+ and 2 mM Mg2+ (white bars) with [Na+]o set at 140, 95, or 50 mM. Increasing the extracellular concentration of Ca2+ and Mg2+ causes a significant reduction of single channel conductance at all concentrations of Na+ examined, but the magnitude of the depression (expressed as a percentage within the white bars) increases as [Na+]o is reduced. In the presence of 0.1 mM Ca2+ and 0.1 mM Mg2+ reducing [Na+]o from 140 to 50 mM, but not to 95 mM, significantly decreases single channel conductance. With Ca2+ and Mg2+ present at 1 and 2 mM, respectively, reducing [Na+]o to both 95 and 50 mM causes significant depression of single channel conductance. Data are the mean ± S.E. of observations made from 3–10 outside-out membrane patches. *, p < 0.05; **, p < 0.01; ***, p < 0.001; as determined by analysis of variance followed by Tukey's post hoc test.

Application of 1 µM 5-HT to cells transfected with the 5-HT_3A(QDA D293A) receptor cDNA elicited only small inward current responses. Hence, we constructed full concentration-response relationships to 5-HT for both the 5-HT_3A(QDA) and 5-HT_3A(QDA D293A) receptors to determine whether the D293A mutation was associated with a reduction in the agonist potency of 5-HT. Such experiments demonstrated that relative to the 5-HT_3A(QDA) receptor (EC₅₀ 1.1 µM), the 5-HT_3A(QDA D293A) construct is less sensitive to 5-HT (EC₅₀ 3.6 µM; supplemental Fig. 1A). Similarly, a comparison of wild-type 5-HT_3A and 5-HT_3A(D293A) receptors revealed that the D293A mutation decreases the potency of 5-HT, as the EC₅₀ value was shifted dextrally from 3 to 7 µM (supplemental Fig. 1B). In subsequent experiments, 2 µM 5-HT was employed to elicit macroscopic currents from cells expressing the 5-HT_3A(D293A) and 5-HT_3A(QDA D293A) receptor constructs.

In common with the 5-HT_3A(QDA) construct, the 5-HT_3A(QDA D293A) mutant maintained exclusive selectivity for cations because the slope of the relationship between the logarithm of (Na⁺)_o and E_5-HT was determined to be 61 mV/decade change in (Na⁺)_o (Fig. 8A). Using solutions E3 and I2 and the voltage ramp protocol, macroscopic currents mediated by the 5-HT_3A(QDA D293A) construct in response to 5-HT demonstrated an E_5-HT of –40.5 ± 0.8 mV (n = 6), corresponding to a P_Ca/P_Cs ratio of only 0.25 ± 0.01 (n = 6) compared with that of 3.7 for the 5-HT_3A(QDA) receptor (Fig. 8B). Similarly, the E_5-HT of –30.6 ± 4.7 mV (n = 6) for the 5-HT_3A(D293A) mutant indicates a P_Ca/P_Cs of 0.44 ± 0.08 (n = 6), which is also significantly reduced versus the wild-type receptor (1.4). The P_Ca/P_Cs ratios of the 5-HT_3A(D293A) and 5-HT_3A(QDA D293A) receptors were not significantly different. The P_Na/P_Cs ratio of either construct was unchanged by the D293A mutation (Fig. 8B). Thus, for both the wild-type 5-HT_3A and 5-HT_3A(QDA) constructs, neutralization of the negatively charged 20' residue greatly reduces the P_Ca/P_Cs ratio but has no effect upon P_Na/P_Cs.

In addition to reducing the relative permeability of Ca²⁺, as evidenced by a decreased sensitivity of E_5-HT to changes in [Ca²⁺]_o (Fig. 8C), the introduction of the D293A mutation into the 5-HT_3A(QDA) receptor construct also decreased single channel conductance. In the presence of 0.1 mM Ca²⁺ and 95 mM Na⁺, the single channel chord conductance of the 5-HT_3A(QDA D293A) receptor was 25.8 ± 1.9 pS (n = 4) at a holding potential of –80 mV in comparison with 41.3 pS for the 5-HT_3A(QDA) construct. Similarly, the single channel chord conductance (at –80 mV) of the 5-HT_3A(QDA D293A) receptor determined with solution E2 (29.8 ± 0.5, n = 7) was significantly less than that of the 5-HT_3A(QDA) construct (40.8 pS). However, no difference in single channel conductance was observed between the 5-HT_3A(QDA) and 5-HT_3A(QDA D293A) constructs over a range of positive holding potentials (60 to 100 mV), indicating that the D293A mutation preferentially suppresses inwardly directed cation flux (Fig. 8D). As found for the 5-HT_3A(QDA) receptor, elevated [Ca²⁺]_o (0.1–30 mM) also depressed the single channel conductance of the 5-HT_3A(QDA D293A) construct in a concentration-dependent manner (Fig. 8E). At all values of [Ca²⁺]_o studied, the single channel chord conductance of the 5-HT_3A(QDA D293A) construct was significantly less than that of the 5-HT_3A(QDA) receptor. Clearly, the alleviation of macroscopic current block by the D293A mutation (34) cannot be attributed to a reduced influence of Ca²⁺ upon single channel conductance.

View larger version (22K): [in this window] [in a new window]   FIGURE 8. Comparison of the relative ion permeability of wild-type, 5-HT3A(D293A), 5-HT3A(QDA), and 5-HT3A(QDA D293A) receptor constructs with Na+ and Ca2+. A, the relationship between the reversal potential (E5-HT) of the macroscopic current response to pressure-applied 5-HT (10 µM) recorded from the 5-HT3A(QDA D293A) receptor construct and the activity of Na+ in the extracellular medium ((Na+)o). Responses were recorded with extracellular electrolytes based on solution E2 wherein the concentration of NaCl was reduced by substitution with sucrose. Intracellular solutions were either CsCl-based I1 (filled circles) or a solution in which NaCl totally replaced CsCl (open circles). The regression line fitted to the filled circles yielded a slope of 61 mV/decade change in (Na+)o. Data points are the mean of 3–6 independent determinations of E5-HT, and vertical bars indicate mean ± S.E. In A, C, D, and E, the dashed gray lines summarize the corresponding data for the 5-HT3A(QDA) receptor construct illustrated in detail in Figs. 3, 4, and 5. B, bar graph illustrating the permeability (Px/PCs) of Na+ (black bars) or Ca2+ (white bars) relative to Cs+ for the indicated receptor constructs. E5-HT was determined with an intracellular CsCl-based solution (I1) as reference and NaCl (146 mM)-based solution E2 or with CaCl2 (100 mM)-based solution E3 as the extracellular electrolyte. Data are the mean ± S.E. of 6–17 independent estimates of Px/PCs. Mutant constructs in which PCa/PCs was significantly different from the wild-type control are indicated with asterisks (p < 0.01; analysis of variance followed by Dunnett's post hoc test). No significant differences in PNa/PCs were found between the wild-type and mutant receptors. C, E5-HT for the 5-HT3A(QDA D293A) receptor construct with [Na+]o set to 95 mM and [Ca2+]o within the range of 0.1 to 30 mM. Note that Ca2+ ion activity ((Ca2+)o) rather than concentration ([Ca2+]o) is plotted. Data are the mean of 3–4 observations. D, current-voltage (i-V) plot for single channel currents recorded from the 5-HT3A(QDA D293A) receptor construct with NaCl-based solution E2 as the extracellular electrolyte and I1 as the intracellular solution. Note that the additional D293A mutation causes an intensification of outward rectification and a reduced single channel conductance at negative potentials in comparison with the 5-HT3A(QDA) construct. Data are the mean of 6–7 observations. E, concentration-response relationship and exemplar single channel currents illustrating the depressant effect of [Ca2+]o upon the single channel conductance of the 5-HT3A(QDA D293A) receptor construct. Note that Ca2+ ion activity, rather than concentration, is plotted. Data are the mean ± S.E. of measurements performed on 3–4 outside-out membrane patches at a holding potential of –80 mV under the ionic conditions described in C.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Our results indicate that the single mutation R436D is sufficient to enhance P_Ca/P_Cs to an extent comparable with that found for the 5-HT_3A(QDA) construct. Analysis of several amino acid substitutions involving neutralization, or inversion, of charge at this key locus suggests that an electrostatic repulsive effect contributes to a suppression of P_Ca/P_Cs in the wild-type receptor. The latter is consistent with the results of modeling studies that place the highly basic (pK_a = 12.48) guanidinium group of Arg-436 within the intracellular permeation pathways (portals) located between adjacent MA-stretch -helices (26, 32). Because of the small maximal width of the portals (26), interactions between partially hydrated permeating ions and Arg-436 are likely.

Previous studies of Ca²⁺ permeation through homomeric 5-HT₃ receptor channels have, because of its very low single channel conductance, been restricted to the analysis of macroscopic currents. The latter are suppressed by extracellular divalent cations by mechanisms that potentially include a reduction in agonist binding affinity (34, 41), an acceleration of the kinetics of deactivation and desensitization (34), and depression of macroscopic and single channel current amplitudes (20, 34), the latter being inferred by fluctuation analysis (20). In the present study, we employed the 5-HT_3A(QDA) receptor construct to examine directly the influence of extracellular Ca²⁺ upon single channel conductance. A solution (E3), in which Ca²⁺ was the sole extracellular cation, supported outwardly rectifying single channel currents but with a chord conductance at negative potentials (5.7 pS), which was greatly reduced in comparison with the value of 40.8 pS obtained with solution E2, in which Na⁺ was the permeant species. Thus, although the permeability of Ca²⁺ relative to Cs⁺ is greatly increased in the 5-HT_3A(QDA) construct, the channel does not conduct Ca²⁺ efficiently. Moreover, the addition of Ca²⁺ to Na⁺-containing extracellular solutions caused a concentration-dependent inhibition of the inwardly directed single channel current amplitude, in a manner reminiscent of that observed for single channel currents mediated by the nicotinic ACh receptor of skeletal muscle (45). Such results are most parsimoniously explained by hypothesizing that the permeation pathway presents a low affinity binding site (or sites) for Ca²⁺, which confers a degree of selectivity for Ca²⁺ over monovalent cations, yet with an off-rate sufficient to allow significant flux of both Na⁺ and Ca²⁺. An interaction between Ca²⁺ and Na⁺ within the channel is supported by our experimental observations indicating the P_Ca/P_Na ratio for the 5-HT_3A(QDA) receptor construct varies with [Ca²⁺]_o (i.e. the principle of independence is violated (39)). Consistent with this notion, the reduction in single channel current amplitude by extracellular divalent cations was more pronounced when [Na⁺]_o was reduced.

We considered the D293A residue of the human 5-HT_3A receptor subunit, which forms the 20' negatively charged ring within the extracellular vestibule of the channel (2, 28), to be a strong candidate for the putative Ca²⁺ binding site that limits the rate of charge transfer through the channel (Fig. 1). Firstly, mutation of the corresponding residue (Asp-298) of the mouse 5-HT_3A subunit to alanine alleviates the block of macroscopic current responses by extracellular Ca²⁺ (34). Secondly, Ca²⁺ reduced the amplitude of single channel currents mediated by the 5-HT_3A(QDA) receptor construct in a voltage-independent manner. The lack of observable voltage dependence suggests a binding site for Ca²⁺ away from the electrical field of the membrane. The 20' residue resides within such a location. However, because Ca²⁺ is permeant, rather than a simple impermeant open channel blocker, it remains possible that Ca²⁺ does interact with a site(s) within the field but that the increased driving force provided by hyperpolarization facilitates its dissociation and inward conduction. Note that this scenario is quite different from that found for an impermeant cationic blocker, for which the dwell time within the channel would be increased by hyperpolarization because of a decreased probability of the molecule exiting the pore to the extracellular environment.

The potency of 5-HT to activate the mouse 5-HT_3A(D298A) construct is less than for the murine wild-type receptor (34), a result concordant with our observation that the D293A mutation introduced into either the human wild-type or 5-HT_3A(QDA) construct causes a dextral shift in the 5-HT concentration-response relationship. The D293A mutation within the 5-HT_3A(QDA) receptor background caused a reduction in the amplitude of inwardly, but not outwardly, directed single channel current events. Such an effect is consistent with a simple electrostatic mechanism in which the negative charge of the five Asp-293 residues acts to increase the local concentration of permeant cations within the extracellular vestibule. Removal of such charges would thus reduce a focusing mechanism, consistent with the appearance of a enhanced outward rectification in the i-V relationship for the 5-HT_3A(QDA D293A) receptor construct versus the 5-HT_3A(QDA) receptor. Very similar effects of neutralizing the extracellular ring of charge upon single channel conductance and rectification have been reported for nicotinic ACh and anion-selective -aminobutyric acid type A and glycine receptors (28). Because of the very low conductance of the wild-type 5-HT_3A receptor (20, 21) and kinetic properties of the 5-HT_3A(D293A) receptor, which are unfavorable to fluctuation analysis (34),⁵ we could not estimate the effect of the D293A mutation upon single channel conductance in this construct. However, it is notable that the pattern of inward rectification of macroscopic currents mediated by the mouse 5-HT_3A(D298A) mutant resembles that of the murine wild-type receptor (34). The latter differs from the present observations comparing the human 5-HT_3A(QDA) and 5-HT_3A(QDA D293A) receptors at the single channel level. An important difference in our studies is the removal of positive electrostatic potential in the intracellular vestibule by the mutations R432Q, R436A, and R440A. Such charges are present in the mouse 5-HT_3A and 5-HT_3A(D298A) receptors studied by Hu and Lovinger (34) and would reduce the local concentration of permeant ions within the intracellular vestibule. The latter would induce a degree of inward rectification. It is possible that such an influence is dominant, masking any tendency toward linearization that might be anticipated from the 5-HT_3A(D293A) mutation. Indeed, in our studies, the macroscopic current I-V relationship shifts from the pattern of inward rectification characteristic of the wild-type 5-HT_3A receptor toward linearity in the 5-HT_3A(QDA) construct.

For both the wild-type 5-HT_3A and the 5-HT_3A(QDA) constructs, the introduction of the D293A mutation drastically reduced the P_Ca/P_Cs ratio. Once more, such an effect can probably be attributed to a reduction in a local negative electrostatic potential that particularly favors the accumulation of Ca²⁺ at the entrance to the pore, as suggested by molecular dynamics simulations performed on models of the ₇ and skeletal muscle nicotinic ACh receptors (46, 47). Such simulations indicate monovalent cations to be stabilized at the 20' position, where the ion dwells during its passage through the channel axis. The greater coulombic attraction between Ca²⁺ and the 20' residue would amplify such interactions. Also, heteromeric ₄β₂ nicotinic ACh receptors, harboring a larger complement of β₂ subunits in which lysine occupies the 20' position, appear to have reduced selectivity toward Ca²⁺ (48). However, the D293A residue does not contribute crucially to cation versus anion selectivity because the P_Na/P_Cl ratio for the 5-HT_3A(QDA D293A) construct was best described as infinite. A previous study of mouse 5-HT_3A receptors in which cysteine r