The single-channel properties of human acetylcholine alpha 7 receptors are altered by fusing alpha 7 to the green fluorescent protein

Abstract

Neuronal nicotinic acetylcholine (AcCho) receptors composed of alpha7-subunits (alpha7-AcChoRs) are involved in many physiological activities. Nevertheless, very little is known about their single-channel characteristics. By using outside-out patch-clamp recordings from Xenopus oocytes expressing wild-type (wt) alpha7-AcChoRs, we identified two classes of channel conductance: a low conductance (gamma(L)) of 72 pS and a high one (gamma(H)) of 87 pS, with mean open-times (tau(op)) of 0.6 ms. The same classes of conductances, but longer tau(op) (3 ms), were seen in experiments with chimeric alpha7 receptors in which the wtalpha7 extracellular C terminus was fused to the green fluorescent protein (wtalpha7-GFP AcChoRs). In contrast, channels with three different conductances were gated by AcCho in oocytes expressing alpha7 receptors carrying a Leu-to-Thr 248 mutation (mutalpha7) or oocytes expressing chimeric mutalpha7-GFP receptors. These conductance levels were significantly smaller, and their mean open-times were larger, than those of wtalpha7-AcChoRs. Interestingly, in the absence of AcCho, these oocytes showed single-channel openings of the same conductances, but shorter tau(op), than those activated by AcCho. Accordingly, human homomeric wtalpha7 receptors open channels of high conductance and brief lifetime, and fusion to GFP lengthens their lifetime. In contrast, mutalpha7 receptors open channels of lower conductance and longer lifetime than those gated by wtalpha7-AcChoRs, and these parameters are not greatly altered by fusing the mutalpha7 to GFP. All this evidence shows that GFP-tagging can alter importantly receptor kinetics, a fact that has to be taken into account whenever tagged proteins are used to study their function.

Figure 1

AcCho-current traces in voltage-clamped oocytes successively used for outside-out patch-clamp recordings. Oocytes were injected with different α7-subunit cDNAs as indicated. Horizontal bars = timing of AcCho applications. AcCho concentrations are as indicated. Note the slowing of AcChoR desensitization in B vs. A and its acceleration in D vs. C.

Figure 2

Time course of the frequency of channel openings from oocytes injected with α7-subunit cDNAs as indicated. Timing of AcCho application and AcCho concentrations as in Fig. 1. (E) Same patch as D. Note the decay of activity during AcCho application in A, the maintained activation throughout AcCho application in C, the activity decay during AcCho application in D, and the sustained channel activity during AcCho application in E.

Figure 3

Single-channel properties of wtα7 receptors. (A) AcCho application to an outside-out patch-clamped membrane from the same oocyte as Fig. 1A, eliciting channel openings. (B) Enlarged single-channel traces from the same record as in A. (C) All-point histogram of amplitudes of the single-channel currents in the same patch. (D) Single-channel I-V relation from the same patch. Points represent mean values ± SE. (A) γ_H = 82 pS. Note current rectification beyond 0 mV. (E) Open time distribution from four outside-out membrane patches best fit by a single exponential component: τ₁ = 0.37 ± 0.02 ms. (F) Closed time distribution from same four patches as E, best fit by a single exponential component: τ₁ = 87 ± 6 ms. No. of channels, 471.

Figure 4

Single-channel properties of wtα7-GFP receptors. (A) AcCho application (bar and dose) to an outside-out patch-clamped membrane from same oocyte as Fig. 1B, eliciting channel openings. (B) Enlarged single-channel traces from the same record as in A. (C) All-point amplitude histogram of the single-channel currents in the same patch. Note small bump at ≈−3 pA corresponding to filtered very short channels illustrated in B and A. (D) Single-channel I-V relation from the same patch. γ_H = 90 pS. Note current rectification beyond 0 mV. (E) Open time distribution from six outside-out membrane patches best fit by two exponential components (with weight): τ₁ = 0.43 ± 0.05 ms (76%) and τ₂ = 9 ± 1 ms (24%). (F) Closed time distribution from same patches as E, best fit by four exponential components: τ₁ = 0.14 ± 0.02 ms (52%), τ₂ = 1.2 ± 0.2 ms (18%), τ₃ = 15 ± 2 ms (17%), and τ₄ = 191 ± 35 ms (12%). No. of channels, 607.

Figure 5

Single-channel properties of mutα7 receptors. (A) AcCho application to an outside-out patch-clamped membrane from same oocyte as Fig. 1C, eliciting channel openings. The open probability increased ≈13-fold (with two channels opening simultaneously) at the beginning of AcCho application. (B) Single-channel traces from the same record as in A. Note two-channel amplitude levels. (C) All-point amplitude histograms of the two-channel populations. (D) Single-channel I-V relations from the same patch. ●, γ_M = 50 pS; ○, γ_L = 42 pS. Note linearity. (E) Open time distribution from four outside-out membrane patches best fit by three exponential components (with weight): τ₁ = 0.2 ± 0.03 ms (54%), τ₂ = 1.8 ± 0.2 ms (41%), and τ₃ = 20 ± 7 ms (5%). (F) Closed time distribution from same patches as E, best fit by the following four exponential components: τ₁ = 0.13 ± 0.03 ms (65%), τ₂ = 0.9 ± 0.6 ms (16%), τ₃ = 9 ± 4 ms (11%), and τ₄ = 62 ± 27 ms (5%). No. of channels, 1,746.

Figure 6

Single-channel properties of mutα7-GFP receptors. (A) AcCho application to an outside-out patch-clamped membrane from same oocyte as Fig. 1D, eliciting channel openings. The open probability increased ≈10-fold after AcCho application. (B) Single-channel traces from the same record as in A. Note two-channel amplitude levels. (C) All-point amplitude histograms of the two-channel populations. (D) Single-channel I-V relations from the same patch. ●, γ_M = 52 pS; ○, γ_L = 40 pS. Note linearity. (E) Open time distribution from four outside-out membrane patches best fit by three exponential components (with weight): τ₁ = 0.18 ± 0.02 ms (83%), τ₂ = 1.2 ± 0.6 ms (9%), and τ₃ = 9 ± 3 ms (8%). (F) Closed time distribution from same patches as E, best fit by the following four exponential components: τ₁ = 0.25 ± 0.05 ms (49%), τ₂ = 1.5 ± 0.4 ms (28%), τ₃ = 9 ± 3 ms (18%), and τ₄ = 76 ± 29 ms (5%). No. of channels, 3,694.