Ion permeation and conduction in a human recombinant 5-HT3 receptor subunit (h5-HT3A)

Abstract

1. A human recombinant homo-oligomeric 5-HT3 receptor (h5-HT3A) expressed in a human embryonic kidney cell line (HEK 293) was characterized using the whole-cell recording configuration of the patch clamp technique. 2. 5-HT evoked transient inward currents (EC50 = 3.4 microM; Hill coefficient = 1.8) that were blocked by the 5-HT3 receptor antagonist ondansetron (IC50 = 103 pM) and by the non-selective agents metoclopramide (IC50 = 69 nM), cocaine (IC50 = 459 nM) and (+)-tubocurarine (IC50 = 2.8 microM). 3. 5-HT-induced currents rectified inwardly and reversed in sign (E5-HT) at a potential of -2.2 mV. N-Methyl-D-glucamine was finitely permeant. Permeability ratios PNa/PCs and PNMDG/PCs were 0.90 and 0.083, respectively. 4. Permeability towards divalent cations was assessed from measurements of E5-HT in media where Ca2+ and Mg2+ replaced Na+. PCa/PCs and PMg/PCs were calculated to be 1.00 and 0.61, respectively. 5. Single channel chord conductance (gamma) estimated from fluctuation analysis of macroscopic currents increased with membrane hyperpolarization from 243 fS at -40 mV to 742 fS at -100 mV. 6. Reducing [Ca2+]o from 2 to 0.1 mM caused an increase in the whole-cell current evoked by 5-HT. A concomitant reduction in [Mg2+]o produced further potentiation. Fluctuation analysis indicates that a voltage-independent augmentation of gamma contributes to this phenomenon. 7. The data indicate that homo-oligomeric receptors composed of h5-HT3A subunits form inwardly rectifying cation-selective ion channels of low conductance that are permeable to Ca2+ and Mg2+.

Figure 1. 5-HT and selective 5-HT₃ receptor agonists evoke inward current responses in HEK 293 cells stably expressing h5-HT_3A receptors

A, traces from one cell depicting the concentration dependence of the peak current response evoked by rapidly superfused 5-HT (0.6-30 μm). Note that the rate of activation and desensitization of the current response increases with agonist concentration. B, inward current responses from one cell comparing the agonist activity of rapidly superfused 5-HT (3 μm) and the 5-HT₃ receptor-selective compounds 2-methyl-5-HT (10 μm), 1-phenylbiguanide (PBG; 80 μm) and m-chlorophenylbiguanide (mCPBG; 1 μm). Currents in A and B were recorded at a holding potential of −60 mV. Horizontal bars above the current traces indicate the period of agonist application. C, graph depicting the concentration dependency of the inward current response evoked by 5-HT (0.3-30 μm). To combine the data from nine different cells, the peak current amplitude evoked by a saturating concentration of 5-HT was assigned a value of 100% and the currents elicited by all other concentrations of agonist are expressed relative to that value. The curve fitted according to eqn (1) yielded an EC₅₀ value of 3.4 ± 0.2 μm and a Hill coefficient of 1.8 ± 0.3 for 5-HT. Vertical bars, where they exceed the size of the symbol, indicate the standard error of the mean.

Figure 2. Inhibition of h5-HT_3A receptor-mediated currents by 5-HT₃ receptor antagonists

Inward current responses evoked by pressure-applied 5-HT (10 μm) were reduced by the bath application of: ondansetron (100 pm) (A), metoclopramide (Met; 100 nm) (B), cocaine (300 nm) (C) and (+)-tubocurarine ((+)-TC; 3 μm) (D). All records are the computer-generated average of 3 consecutive responses to 5-HT recorded at a holding potential of −60 mV. Each antagonist was tested on a different cell. E, graphical depiction of the concentration-dependent inhibition of the 5-HT-evoked current by 5-HT₃ receptor antagonists. Peak current amplitude is expressed as a percentage of that observed in the absence of antagonist. The fitted curves (eqn (1)) give IC₅₀ values of: ondansetron (▪; 103 pm), metoclopramide (•; 69 nm), cocaine (▾; 459 nm) and (+)-tubocurarine (♦; 2.2 μm). Each data point is the mean of 5–6 experiments conducted upon different cells. Vertical bars indicate the standard error of the mean.

Figure 3. Ionic dependency of h5-HT_3A receptor-mediated currents

A, comparison of I-V relationships determined by a voltage ramp (−150 to +50 mV; 0.25 mV ms⁻¹), applied at the peak of the current response to 5-HT (1 μm; curve), and peak current responses evoked by pressure-applied 5-HT (10 μm; ▪) recorded at steady holding potentials in the range −100 to +40 mV from a single cell. The relationships were superimposed by applying a constant scaling factor to all currents evoked by pressure-applied 5-HT. Currents were recorded in the presence of the Na⁺-based extracellular solution E2 and a nominally Mg²⁺-free Cs⁺-containing pipette solution. E_5-HT was estimated to be approximately 0 mV. Leakage currents determined in the absence of 5-HT have been subtracted. B, I-V relationship of the 5-HT (1 μm)-induced current determined by voltage ramp from a single cell exposed to extracellular media containing 143 mm (1), 75 mm (2) or 20 mm Na⁺ (3). Equimolar substitution of Na⁺ was performed with NMDG. The inset displays portions of the I-V relationships on expanded voltage and current axes to clarify the potential at which there is zero net current (i.e. E_5-HT). E_5-HT in the exemplar cell was −2 mV in 143 mm Na⁺, −14 mV in 75 mm Na⁺ and −46 mV in 20 mm Na⁺. Leakage currents determined in the appropriate solutions were subtracted. C and D, I-V relationships for 5-HT-evoked currents obtained from cells bathed in extracellular media in which either Ca²⁺ (100 mm; C) or Mg²⁺ (100 mm; D) was totally replaced Na⁺. E_5-HT was estimated to be −10 and −20 mV, respectively.

Figure 4. Extracellular divalent cations suppress h5-HT_3A receptor-mediated currents

I-V relationships, obtained from one cell by voltage ramps (−150 to +50 mV; 0.25 mV ms⁻¹), applied at the peak of the current response to 5-HT (1 μm) in Na⁺-based extracellular media containing 2 mm Ca²⁺ and 2 mm Mg²⁺ (1), 2 mm Mg²⁺ and 0.1 mm Ca²⁺ (2), or 0.1 mm Ca²⁺ and 0.1 mm Mg²⁺ (3). Note that inward rectification of the 5-HT-induced current is observed under all conditions and that suppression of the current by extracellular divalent cations appears to be voltage independent. The inset shows that E_5-HT is insensitive to the small changes in either [Ca²⁺]_o or [Mg²⁺]_o.

Figure 5. Fluctuation analysis whole-cell currents mediated by h5-HT_3A receptors

Examples from a single cell of low gain DC-coupled records (top panel) and high gain AC-coupled (1–1000 Hz bandwidth) records (middle panel) of inward current responses evoked by bath-applied 5-HT (1 μm; horizontal bar) at holding potentials of −40 (A), −60 (B), −80 (C) and −100 mV (D) in the presence of standard extracellular (E1) and intracellular media (I1). Note that the development and desensitization of the inward current response are paralleled by respective increases and decreases in membrane current fluctuations. The latter are most pronounced at hyperpolarized potentials. The lower panels illustrate the relationship between the variance of the AC-coupled current and DC amplitude throughout the 5-HT-induced response. The slope of the line fitted to the data points, by linear regression analysis, provides estimates of single channel current amplitudes of 5.9, 12.0, 31.4 and 61.1 fA at holding potentials of −40, −60, −80 and −100 mV, respectively. Background current variance recorded in the absence of 5-HT at each holding potential was subtracted.

Figure 6. Extracellular divalent cations decrease the single channel conductance of h5-HT_3A receptors

A, graph depicting the influence of [Ca²⁺]_o and [Mg²⁺]_o upon the single channel chord conductance of h5-HT_3A estimated from fluctuation analysis of macroscopic current responses recorded in the presence of 2 mm Ca²⁺-2 mm Mg²⁺ (▪); 0.1 mm Ca²⁺-2 mm Mg²⁺ (▴) or 0.1 mm Ca²⁺- 0.1 mm Mg²⁺ (♦). Inward rectification is apparent under all conditions and is little affected by the extracellular concentrations of divalent cations. Data points are the means of 4–7 determinations of single channel chord conductance performed on different cells. Vertical bars indicate the standard error of the mean. B, C and D, comparison of the voltage dependence of macroscopic and single channel currents evoked by 5-HT in 3 different cells bathed in media containing 2 mm Ca²⁺ and 2 mm Mg²⁺ (B); 2 mm Mg²⁺ and 0.1 mm Ca²⁺ (C) or 0.1 mm Ca²⁺ and 0.1 mm Mg²⁺ (D). The I-V relationship of the macroscopic current response to 5-HT (1 μm) was determined by a voltage ramp (−150 to +50 mV; 0.25 mV ms⁻¹), applied at the peak of the current response to 5-HT (1 μm), whereas single channel current amplitudes were estimated by stationary fluctuation analysis at holding potentials of −40, −60, −80 and −100 mV. Superimposition of the macroscopic and single channel current amplitudes was achieved by scaling the latter by a constant factor. The data suggest that, at least within the voltage range −40 to −100 mV, inward rectification under each ionic condition examined can be attributed to the conductance properties of the h5-HT_3A receptor channel.