Human alpha4beta2 neuronal nicotinic acetylcholine receptor in HEK 293 cells: A patch-clamp study

Abstract

The cloning and expression of genes encoding for the human neuronal nicotinic acetylcholine receptors (nAChRs) has opened new possibilities for investigating their physiological and pharmacological properties. Cells (HEK 293) stably transfected with two of the major brain subunits, alpha4 and beta2, were characterized electrophysiologically using the patch-clamp technique. Fast application of the natural ligand ACh can evoke currents up to 3500 pA, with an apparent affinity (EC50) of 3 microM and a Hill coefficient of 1.2. The rank order of potency of four nAChR ligands to activate human alpha4beta2 receptors is (-)-nicotine > ACh > (-)-cytisine > ABT-418. At saturating concentrations, the efficacy of these ligands is ABT-418 >> (-)-nicotine > ACh >> (-)-cytisine > GTS-21 (previously named DMXB). Coapplication of 1 microM ACh with known nAChR inhibitors such as dihydro-beta-erythroidine and methyllycaconitine reversibly reduces the current evoked by the agonist with respective IC50 values of 80 nM and 1.5 microM. The current-voltage relationship of human alpha4beta2 displays a strong rectification at positive potentials. Experiments of ionic substitutions suggest that human alpha4beta2 nAChRs are permeable to sodium and potassium ions. In the "outside-out" configuration, ACh evokes unitary currents (main conductance 46 pS) characterized by a very fast rundown. Potentiation of the ACh-evoked currents is observed when the extracellular calcium concentration is increased from 0.2 to 2 mM. In contrast, however, a reduction of the evoked currents is observed when calcium concentration is elevated above 2 mM.

Fig. 1.

Fast drug application using a multibarrel puffer. The time course for a complete solution exchange on a cell was determined by performing a sodium jump during a steady application of a low ACh concentration. The first “control” current (bold trace) was recorded in a standard medium containing 130 mm NaCl. A second current (light trace) was elicited by applying ACh in a solution containing 65 mmNaCl + 130 mm mannitol. Then the solution was switched for the standard medium (rectangle with diagonal lines), and the current was returned to the level of the control trace. The inset (bottom) presents with an expanded time scale the current rise time induced by the sodium jump. Complete solution exchange is performed within 20 msec.

Fig. 2.

Sensitivity of human α4β2 nAChRs toward ACh and (−)-nicotine. A, Currents evoked by increasing concentrations of ACh (pulse of 500 msec) in a cell held at −100 mV.B, Concentration–response curves for (−)-nicotine and ACh. Dose–response relationships were fitted with the empirical Hill equation y = 1/(1 + ((EC₅₀/[agonist])ⁿ)). Half-effective concentrations (EC₅₀) were then determined: 1.6 μm (n = 1.3) for (−)-nicotine (5 cells) and 3 μm (n = 1.2) for ACh (19 cells).

Fig. 3.

Agonists are characterized by different affinities and efficacies for human α4β2 nAChRs. A, Transfected HEK 293 cells were challenged with six to seven concentrations of each agonist (ranging from 0.3 to 300 μm). Data were then normalized against the largest evoked current and plotted on a semi-logarithm scale. Mean values were fitted with the empirical Hill equation (see Fig. 2 legend) giving EC₅₀ values (μm) and Hill coefficient (n): (−)-cytisine (11.6, 1.5; 7 cells) and ABT-418 (13.9, 1.3; 13 cells).B, The efficacy of the agonist was determined at saturating concentration (100 μm each but 300 μm for ABT-418), with a holding potential of −100 mV. For accurate determination, the following protocol of sequential drug application (500 msec each, every 4.5 sec) was used: alternate applications of the agonists to be tested and ACh. A first set of measures was performed with ACh, (−)-nicotine, and ABT-418 (7 cells) and another one with ACh, (−)-cytisine and GTS-21 (7 cells). Currents were normalized as in A. ABT-418 evoked 190 ± 15% of saturating ACh current, whereas (−)-nicotine produced 112 ± 7%. In contrast, (−)-cytisine and GTS-21 can elicit only 16 ± 2% and 6 ± 2% of the ACh-evoked current, respectively. A typical example of the currents evoked on one cell by 100 μm ACh, (−)-nicotine and 100 μm ABT-418 is presented in C. A very weak current evoked by 100 μm GTS-21 is compared with the 100 μm ACh current for another cell in D.

Fig. 4.

Dihydro-β-erythroidine (DHβE) antagonizes the effect of ACh on the human α4β2 nAChRs. Every 10 sec, a 1200 msec pulse was delivered alternately with 1 μm ACh alone or with 1 μm ACh and increasing concentrations of DHβE (0–600 nm) in co-application for the last 800 msec (see an example of the recorded currents on one cell in the inset). The full block of the ACh-evoked current by 600 nm DHβE was totally reversed within 10 sec (not shown; 3 cells). The inhibitory effect of DHβE was measured at the end of the pulse of co-application (steady-state current) and normalized toward the value of the plateau amplitude of the preceding ACh-evoked current. Values were plotted against the concentrations of DHβE (on a logarithm scale) and fitted with the empirical Hill equation: y = 1/(1 + (([DHβE]/IC₅₀)ⁿ)), where IC₅₀ and n represent the half-inhibitory concentration and the Hill coefficient, respectively. The calculated IC₅₀ value is 80 nm with an nvalue of 1.1 (n = 6).

Fig. 5.

Methyllycaconitine (MLA) inhibits the ACh-evoked current at the human α4β2 nAChR in the micromolar range. A protocol identical to that used in Figure 4 was applied for the determination of the antagonistic properties of MLA. Traces corresponding to a 1 μm ACh-evoked current before and after a 6 μm MLA jump are presented in A. Normalized values were plotted as a function of the concentration of MLA represented on a logarithm scale (B) and fitted with the empirical Hill equation (see Fig. 4 legend). An IC₅₀ of 1.5 μm (n = 1.8) was calculated from the mean ± SEM values collected on four cells.

Fig. 6.

Human α4β2 nAChRs demonstrate strong rectification at positive potentials and are highly permeable to Na⁺ and K⁺ ions. ACh-evoked currents were recorded for holding potentials ranging from −100 to 40 mV with 10 mV steps every 4.5 sec in the standard extracellular solution; ACh was delivered 400 msec after the new set of the voltage. Apresents typical currents; for clarity, traces were omitted for all odd values. B, Currents were normalized against values measured at −100 mV. Mean values of three cells were plotted as a function of the holding potentials. Data were fit (solid line) by the following equation: y = (V − E_r)/(1 + exp(α × (V − V_1/2))), whereV is the holding voltage, E_ris the current reversal potential, V_1/2 is the potential for the half-current amplitude, and α is the slope factor. This formula corresponds to the product of the driving force and a Boltzmann equation. Optimal fit was obtained withV_1/2 = −83.1 mV and α = 0.043. For the determination of the selectivity against cations, experiments using voltage ramps were performed with different extracellular solutions (C, D). The membrane was held at −10 mV, and a first ramp (from 40 to −140 mV in 400 msec) was applied in saline medium (without ACh) for the determination of the leak current. Four seconds later, 1 μm ACh was delivered for 800 msec, and the voltage ramp was applied again 200 msec after the beginning of ACh delivery. The leak current has been subtracted to the ACh-evoked current for all the data presented. For clarity, experimental current values are plotted every 20 points, corresponding to approximately one measurement every 7.5 mV. When extracellular Na⁺ chloride (NaCl) was replaced by mannitol, the quantity of ACh-evoked current was very weak (C), indicating a high permeability of the human α4β2 nAChR for Na⁺ ions (but see text). This nAChR is also highly permeable to potassium ions. When extracellular Na⁺ chloride is replaced by an equimolar concentration of potassium chloride, the amplitude of the ACh-evoked current is still large (D); note that the reversal potential for potassium ions switched from approximately −80 mV to 0 mV during this ionic substitution.

Fig. 7.

In the outside-out configuration, ACh-evoked single channels are blocked by DHβE, display a main conductance of 46 pS, and do not rectify at positive holding voltages. Intracellular solution (see Materials and Methods) contained 2 mmMgCl₂. A high concentration of DHβE (600 nm; IC₅₀ = 80 nm) completely inhibited the ACh-evoked single channels (A), demonstrating that these single-channel currents resulted from nAChR activation by ACh (n = 3). An outside-out patch stimulated by ACh at different holding potential clearly showed the voltage dependence of the ACh-evoked current and the predominant conductance observed in many other records (n = 46) (B). This patch contained at least two channels, but their activity disappeared before we were able to record at positive potentials. The closed (c) and opened (o) states are indicated by the dotted lines. In contrast to the whole-cell configuration experiments, the current–voltage relationship for the main conductance of the human α4β2 nAChR is linear and shows no rectification (C). The amplitude values collected for six outside-out patches were fitted (straight line) with the current equation i = γ × (E − E_r), where γ is the conductance, E_r is the reversal potential, and E is the holding potential. The human α4β2 nAChR displays a main conductance of 46 pS with a reversal potential of −6.8 mV, confirming the high permeability of this ligand-gated channel for Na⁺ and K⁺ ions.X corresponds to single-channel amplitudes measured on a patch lasting long enough to allow examination of single-channel apertures at positive voltages. D, Current traces recorded at −100 and 60 mV. E, Loss of rectification in the outside-out configuration was also observed on large membrane patches containing numerous receptor proteins. Traces were recorded at −60 and 40 mV, respectively. In D–E, currents were elicited by 1 μm ACh (horizontal bar) applications.

Fig. 8.

ACh-elicited single channels present fast run-down properties. In the outside-out configuration, the activity of the channels disappeared within minutes. The current recorded on a typical patch is presented in A, and the corresponding peak current amplitudes were plotted as a function of time inB. The current–time values were fitted with a single exponential function giving a time constant of 46.8 sec.

Fig. 9.

High external Ca²⁺ decreases both whole-cell and single-channel currents of the human α4β2 nAChR. Voltage ramps (see Fig. 6 legend) were performed in external solutions containing (in mm): 130 NaCl, 10 HEPES, 20 glucose, and varying concentrations of CaCl₂. When external calcium is increased from 0 to 2 mm, a significant potentiation of the ACh-evoked current for holding potentials less than −50 mV was observed; however, high calcium concentrations (10 or 20 mm) strongly inhibit the ACh-evoked current for holding potentials below 0 mV. A displays typical current–voltage relationships recorded on a single cell in three different calcium concentrations (1 μm ACh). For clarity, the current value is plotted once every 20 recorded points, corresponding to approximately one measurement every 7.5 mV. Continuous lines were computed using the same equation as for Figure 6. The calcium effects have been quantified by measuring for each cell the amplitude of the plateau currents (elicited by 1 μm ACh, 500 msec) recorded in three different calcium concentrations at −100 mV (B). The mean amplitude of the currents recorded in 0.2 mm calcium represents 47.8 ± 4.1% of the mean current measured in 2 mm calcium, whereas the mean current recorded in 20 mm calcium represents only 35.8 ± 4.3% (5 cells). High extracellular calcium is a negative modulator of the human α4β2 nAChR (C). When external Ca²⁺ jumped to 10 and 20 mm during the ACh pulse, a fast and fully reversible block of the ACh-evoked current was observed, indicating the concentration dependence of the calcium effect (n = 3). A high extracellular calcium concentration decreases the single-channel conductance of the human α4β2 nAChR (D–F). Holding potentials of −100, −80, −60, −40, −20, and 20 mV were changed at 5 sec intervals. One second after the voltage setting, the patch was challenged by 3 μm ACh for 200 msec. Such protocol was applied alternately in 20 and 2 mm CaCl₂ (3 patches).D presents two traces of the records performed successively in 20 and 2 mm CaCl₂. Even in the presence of the fast run-down mechanism, the total amount of current is much larger in 2 than in 20 mm Ca²⁺ (at all membrane potentials tested). The horizontal barrepresents the 3 μm ACh pulse. Reduction of the single-channel conductance induced by high extracellular calcium is illustrated in E. Patch was held throughout the experiment at −100 mV. Single-channel amplitudes (corresponding to the main conductance observed) were plotted as a function of the holding potential (F) and fit with the equationi = γ × (E −E_r), where i is the current, γ is the conductance, E is the holding voltage, andE_r is the current reversal potential. In 20 mm Ca²⁺, the main conductance decreases from 46 to 28 pS (see text).