Neuronal nicotinic acetylcholine receptors are blocked by intracellular spermine in a voltage-dependent manner

Abstract

A common feature of neuronal nicotinic acetylcholine receptors (nAChRs) is that they conduct inward current at negative membrane potentials but little outward current at positive membrane potentials, a property referred to as inward rectification. Physiologically, inward rectification serves important functions, and the main goal of our study was to investigate the mechanisms underlying the rectification of these receptors. We examined recombinant alpha3beta4 and alpha4beta2 neuronal nAChR subtypes expressed in Xenopus oocytes and native nAChRs expressed on superior cervical ganglion (SCG) neurons. Whole-cell ACh-evoked currents recorded from these receptors exhibited strong inward rectification. In contrast, we showed that single-channel currents from these neuronal nAChRs measured in outside-out patches outwardly rectify. On the basis of recent findings that spermine, a ubiquitous intracellular polyamine, confers rectification to glutamate receptors and inwardly rectifying potassium channels, we investigated whether spermine causes neuronal nAChRs to inwardly rectify. When spermine was added to the patch electrode in outside-out recordings, it caused a concentration- and voltage-dependent block of ACh-evoked single-channel currents. Using these single-channel data and physiological concentrations of intracellular spermine, we could account for the inward rectification of macroscopic whole-cell ACh-evoked conductance-voltage relationships. Therefore, we conclude that the voltage-dependent block by intracellular spermine underlies inward rectification of neuronal nAChRs. We also found that extracellular spermine blocks both alpha3beta4 and alpha4beta2 receptors; this finding points to a mechanism whereby increases in extracellular spermine, perhaps during pathological conditions, could selectively block these receptors.

Fig. 1.

Macroscopic ACh-evoked currents recorded from SCG neurons show strong inward rectification. A, Whole-cell currents were recorded from an SCG neuron while the membrane potential was ramped from −60 to +50 mV (333 mV/sec). The top trace shows the response of the neuron in the absence of agonist, and the bottom trace shows the response to 100 μm ACh. B, This figure shows the current–voltage (I–V) relationship for the neuron in A. The whole-cellI–V curve was obtained by subtracting the current in control solution from the current in the presence of ACh, during the voltage ramp. The I–Vplot shows strong inward rectification.

Fig. 2.

Whole-cell ACh-evoked current–voltage relationships for recombinant α₃β₄, α₄β₂, and α₄β₂E260 expressed inXenopus oocytes show strong inward rectification.A, C, and E show whole-cell I–V curves for three oocytes expressing recombinant α₃β₄, α₄β₂, and α₄β₂E260, respectively. Macroscopic currents were recorded in response to voltage ramps from −80 to +50 mV (333 mV/sec) in the absence or presence of ACh (1 μm for α₄β₂ and α₄β₂E260, and 20 μm for α₃β₄), and the net currents were obtained as explained in Figure 1. Note: the strong inward rectification of the I–V curve is similar to that of whole-cell I–V in SCG neurons (Fig. 1B). B,D, and F show the corresponding conductance–voltage relationships (G–Vplot) for α₃β₄, α₄β₂, and α₄β₂E260, respectively. Conductance at each holding potential is normalized to the conductance at −80 mV.

Fig. 3.

Outside-out single-channel currents fromXenopus oocytes expressing α₃β₄, α₄β₂, and α₄β₂E260 do not show inward rectification.A, C, and E show steady-state single-channel recordings from outside-out patches of oocytes expressing α₃β₄, α₄β₂, or α₄β₂E260, respectively. These recordings were performed in the continuous presence of ACh: 100–200 nm for α₄β₂ and α₄β₂E260 and 1–2 μm for α₃β₄. Numbers on theleft of each trace correspond to the holding potential at which that recording was obtained. The dotted linesmark the zero current level for each trace. B,D, and F show the single-channelI–V plots for α₃β₄, α₄β₂, or α₄β₂E260, respectively. TheseI–V plots, in contrast to the corresponding macroscopic I–V plots (Fig. 2A,C,E), show a slight outward rectification. Each point in the plots represents the mean ± SE of single-channel current amplitudes from four patches for α₃β₄, eight patches for α₄β₂, and eight patches for α₄β₂E260.

Fig. 4.

Voltage-dependence at the single-channel level cannot account for the macroscopic inward rectification.A, Representative trace showing α₄β₂ single-channel recordings from an outside-out patch while V_m was stepped between −50 and +50 mV (1 sec at eachV_m). Dotted lines mark the zero current level. B, This trace shows currents recorded from an outside-out macropatch expressing α₄β₂ nAChR subtypes. The current was recorded in response to 200 nm ACh whileV_m was ramped from −85 to +85 mV at 1 V/sec. The I–V curve was obtained by subtracting the current after run-down of single-channel activity from the current obtained 30 sec after excision of the patch.C, This trace shows two superimposed α₄β₂ single-channel recordings from an outside-out patch while the V_m was ramped from −100 to +90 mV (1 V/sec). These traces were obtained in the continuous presence of 100 nm ACh. D, This figure shows single-channel currents from an outside-out patch expressing α₄β₂E260 (ACh = 200 nm) while V_m was ramped from −100 to +90 mV (1 V/sec). For this patch, 1 mmMg²⁺ had been added to the recording pipette. Note: outward single-channel openings at positive membrane potentials are smaller than those in control recordings, suggesting a moderate block by intracellular Mg²⁺.

Fig. 5.

Spermine causes inward rectification of ACh-evoked currents in outside-out patches. A, These traces are outside-out single-channel recordings (100 nm ACh) at different holding potentials from an oocyte expressing α₄β₂E260 receptors with 33 μmspermine added to the intracellular pipette solution. Dotted lines mark the zero current level. B, This figure shows outside-out recordings (ACh = 100 nm) from an oocyte expressing α₄β₂ receptors with 1 μm spermine added to the recording electrode; the outward α₄β₂ single-channel currents at +60 mV are reduced by ∼40%. Dotted lines mark the first open channel level for each trace. C, This figure shows the steady-state single-channel I–Vrelationship for α₄β₂ receptors in the absence (▪; n = 12) or in the presence of 1 μm (▴; n = 4), 10 μm(•; n = 6), or 100 μm (▾;n = 4) spermine in the recording electrode.I–V plots show progressive inward rectification of single-channel current with increasing concentrations of intracellular spermine. Solid lines are polynomial fits.

Fig. 6.

Analysis of voltage- and concentration-dependent block by intracellular spermine. A, This figure shows single-channel G–V relationships for α₄β₂ in the absence or the presence of 1, 3.3, 10, 33, 50, or 100 μm spermine in the recording electrode. Single-channel conductance in control patches increased with depolarization (n = 14), whereas single-channel conductance in the presence of spermine progressively decreased with depolarization. Increasing the concentration of spermine causes a leftward shift of theG–V relationship. Solid lines are fits to data points using Equation 1. Points represent the single-channel conductance in the presence of spermine as a fraction of the control conductance for each holding potential; all points are normalized to the conductance at −100 mV. Data are obtained from three to six outside-out patches for each spermine concentration (mean ± SE). Dotted lines are polynomial fits to the data. B, This figure shows the concentration–response relationship for spermine at different holding potentials. Affinity of spermine for α₄β₂receptor is increased with depolarization. Data points are derived fromA, and solid lines are fits using Equation 1. The dotted line is the theoretical fit at 0 mV predicted by Equation 1. C, This figure shows a representative G–V plot of the macroscopic ACh-evoked current recorded from an oocyte expressing α₄β₂ receptors. We fit thisG–V curve with Equation 1, using theK_d(0) and zδ values obtained from single-channel analysis (solid line).

Fig. 7.

Intracellular spermine blocks native SCG nAChRs at the single-channel level. A, This figure shows single-channel recordings obtained from an SCG neuron in an outside-out patch; native SCG nAChRs show both inward and outward currents in response to 20 μm ACh. Dotted lines mark the zero current and the first open state. B, Outside-out single-channel recordings from another SCG neuron with 100 μm spermine added to the recording electrode. Spermine (100 μm) completely blocked the outward single-channel openings and reduced the amplitude of the inward openings ([ACh] = 20 μm). At positive potentials, dotted linesmark the baseline, and at negative potentials, dotted lines mark the baseline and the first open state.C, This figure shows ACh-evoked single-channelI–V curves obtained from outside-out patches from SCG neurons. The controlI–V curve (○; n = 7) exhibits slight outward rectification, similar to recombinant neuronal nAChRs (Fig. 3B,D,F). In the presence of 100 μm spermine in the recording electrode (•;n = 4), however, theI–V relationship exhibits strong inward rectification. Single-channel amplitudes were measured in both steady-state and ramp experiments, and points represent mean ± SE. Solid lines are polynomial fits. D, This figure shows the current in response to 20 μm ACh recorded from an outside-out macropatch from an SCG neuron in the presence of 100 μm spermine in the recording electrode. The current was recorded while V_m was ramped from −60 to +50 mV (at 1V/sec). The solid line is a polynomial fit.

Fig. 8.

Block by intracellular spermine underlies the inward rectification of macroscopic ACh-evoked currents in SCG neurons.A, This figure shows the single-channelG–V plot for native SCG nAChRs in the presence of 100 μm spermine. The single-channel conductance shows a progressive decrease with depolarization. Points represent the single-channel cord conductance (mean ± SE;n = 4) in the presence of spermine as a fraction of the control single-channel conductance at every potential; all values were normalized to the conductance at −75 mV. The solid line is the fit to the data using Equation 1. B, This figure represents the whole-cellG–V plot obtained from an SCG neuron. Similar to the single-channel conductance in the presence of spermine, the macroscopic ACh-evoked conductance in SCG neurons shows a progressive decrease with depolarization. The solid lineis a fit to these data using Equation 1. This fit was performed using values for K_d(0) and zδ from single-channel analysis (see Results).

Fig. 9.

Extracellular spermine blocks macroscopic ACh-evoked inward currents. A, This figure shows inward ACh-evoked currents recorded from an oocyte expressing α₄β₂ in the absence and the presence of extracellular spermine. At −90 mV, 33 μm extracellular spermine blocks the current in response to 1 μm ACh by 45%. B, Macroscopic I–Vrelationship for the oocyte in A is plotted in the presence and absence of 33 μm extracellular spermine. Inward rectification is not affected by extracellular spermine.C, This figure shows the change in the macroscopic conductance versus the membrane potential in the presence of 33 μm extracellular spermine. The conductance in the presence of spermine is shown as a fraction of the control conductance at every membrane potential and then normalized to the conductance at −90 mV. D, This figure shows the inhibition curve for α₄β₂ receptors with increasing concentrations of extracellular spermine. Extracellular spermine blocks the ACh-evoked current in α₄β₂-expressing oocytes in a concentration-dependent manner. Each spermine concentration was tested on at least seven oocytes (mean ± SE).Solid line is a fit for a single binding site isotherm (see Results).