Structural elements near the C-terminus are responsible for changes in nicotinic receptor gating kinetics following patch excision

Abstract

We have studied the effect of patch excision on the gating kinetics of muscle nicotinic acetylcholine receptors transiently expressed in HEK 293 cells. The experiments were performed on embryonic and adult wild-type, and several mutated, receptors using acetylcholine, carbamylcholine and tetramethylammonium as agonists. We show that patch excision of cell-attached patches into the inside-out configuration led to a reduction of mean open duration in receptors containing a gamma-subunit (embryonic) but not an epsilon-subunit (adult receptors). Kinetic analysis of an embryonic receptor containing a mutated residue, alphaY93F, showed that the reduction in the mean open duration upon patch excision was mainly caused by an increase in the channel closing rate constant. This was confirmed by experiments on embryonic wild-type receptors using carbamylcholine as an agonist with low efficacy. By expressing receptors containing chimeric gamma-epsilon subunits we found that segments of the gamma-subunit corresponding to a region within the M3-M4 linker (the amphipathic helix, HA) and the M4 transmembrane domain were required for the reduction in channel open duration after excision. The results indicate that particular residues in both M4 and HA are required to allow the change in open time after excision. This finding suggests that there is an interaction between these two regions in determining the modulation of gating kinetics.

Figure 5. The aligned sequences of the constructs

The portion of the sequences shown begins near the start of the HA region (indicated above the top line) and ends in the M4 region. The sequences starting at position 416 (γ-subunit, top line) or position 398 (ɛ-subunit, bottom line) are shown. Residues derived from the γ-subunit are shown in bold and underlined. The construct is identified in the left-most column. The second column gives the source of the sequence preceeding and following the portion shown (the backbone of the construct) – for example χ9 has residues from the γ-subunit before and after the region shown, whereas χGINE has residues from the ɛ-subunit.

(Model 1)

Figure 1. The effect of patch excision on single-channel activity from embryonic wild-type receptors

A, single-channel openings elicited by 5 μm ACh in cell-attached and inside-out configurations, and their respective open interval duration histograms (different patches; 1899 and 1459, events, respectively). B, single-channel clusters elicited by 100 μm ACh in cell-attached and inside-out configurations, and their respective closed and open interval duration histograms (different patches; 1730 and 3586 events, respectively). The membrane potential was −70 mV; inward current is downward. Open time histograms were fitted with a single exponential, and the time constants are given in the figure. The closed time histograms were fitted with the sum of two exponentials, and the time constants are given in the figure.

Figure 2. The effect of patch excision on single-channel activity from adult wild-type receptors

A, single-channel openings elicited by 5 μm ACh in cell-attached and inside-out configurations, and their respective open interval duration histograms (different patches; 694 and 440 events, respectively). B, single-channel openings elicited by 50 μm CCh in cell-attached and inside-out configurations, and their respective open interval duration histograms (different patches; 703 and 664 events, respectively). The membrane potential was −70 mV; inward current is downward. The histograms were fitted with a single exponential, and the time constants are given in the figure.

Figure 6. Summary of the effects of patch excision on the open duration of single-channel openings measured at membrane potentials between −80 and −60 mV

The change in the apparent open duration (shown as log effect) is calculated as the mean effect from 2–6 experiments (±s.d.). The columns show data from the following constructs and agonists: embryonic receptors (1–3): 1, αβγδ (ACh); 2, αβγδ (CCh); 3, αY93Fβγδ (ACh); adult receptors (4–7): 4, αβδɛ (ACh); 5, αβδɛ (CCh); 6, αβδɛ (TMA); 7, αY93Fβδɛ (ACh); chimeric receptors (8–12): 8, αY93Fβδχ9 (ACh); 9, αβδχ19 (TMA); 10, αβδχ32 (TMA); 11, αβδχGINE (TMA); 12, αβδγWA (TMA). For all agonists, non-desensitizing concentrations were used (see Methods). The effects of patch excision were statistically significant (P < 0.05) in lanes 1, 2, 3 and 11.

Figure 3. The effect of patch excision on single-channel activity from Y93Fβγδ receptors

A, single-channel openings elicited by 50 μm ACh in cell-attached and inside-out configurations, and their respective open interval duration histograms (different patches; 422 and 389 events, respectively). B, single-channel clusters elicited by 8 mm ACh in cell-attached and inside-out configurations, and their respective closed and open interval duration histograms (different patches; 10606 and 3920 events, respectively). The membrane potential was −70 mV; inward current is downward. The closed and open time histograms were fitted with a single exponential, and the time constants are given in the figure.

Figure 4. Concentration-response parameters for the αY93Fβγδ and αY93Fβδɛ mutant receptors

A, effective opening rate, effective closing rate and P_o for αY93Fβγδ receptors as a function of [ACh]. Open symbols are for cell-attached, filled symbols for inside-out patches. Each symbol shows data from one patch. Continuous lines are from fits to the Hill equation. For cell-attached patches: β = 1016 s⁻¹, α’ = 401 s⁻¹, P_o,max = 0.76, EC₅₀ = 631 μm, n = 1.3. For inside-out patches: β = 1078 s⁻¹, α’ = 822 s⁻¹, P_o,max = 0.55, EC₅₀ = 701 μm, n = 2.2. B, effective opening, effective closing rate and P_o of αY93Fβδɛ as a function of [ACh]. For cell-attached patches: β = 1220 s⁻¹, α’ = 1708 s⁻¹, P_o,max = 0.38, EC₅₀ = 1145 μm, n = 1.8. For inside-out patches: β = 1097 s⁻¹, α’ = 1866 s⁻¹, P_o,max = 0.35, EC₅₀ = 1424 μm, n = 1.6.

Figure 7. The relationship between mean open interval duration and amplitude of single-channel openings elicited by 5–50 μm CCh

Circles: embryonic wild-type receptors (○, cell attached; •, inside-out); squares: adult wild-type receptors (□, cell attached, ▪, inside-out). The data were fitted using eqn (1). For embryonic receptors, τ₀ = 1.8 ms and f = 0.33 for the cell-attached configuration, τ₀ = 0.8 ms and f = 0.31 for the inside-out configuration. For adult receptors, τ₀ = 0.19 ms and f = 0.29 for the cell-attached configuration, τ₀ = 0.16 ms and f = 0.33 for the inside-out configuration.