The minimum M3-M4 loop length of neurotransmitter-activated pentameric receptors is critical for the structural integrity of cytoplasmic portals

Abstract

The 5-HT3A receptor homology model, based on the partial structure of the nicotinic acetylcholine receptor from Torpedo marmorata, reveals an asymmetric ion channel with five portals framed by adjacent helical amphipathic (HA) stretches within the 114-residue loop between the M3 and M4 membrane-spanning domains. The positive charge of Arg-436, located within the HA stretch, is a rate-limiting determinant of single channel conductance (γ). Further analysis reveals that positive charge and volume of residue 436 are determinants of 5-HT3A receptor inward rectification, exposing an additional role for portals. A structurally unresolved stretch of 85 residues constitutes the bulk of the M3-M4 loop, leaving a >45-Å gap in the model between M3 and the HA stretch. There are no additional structural data for this loop, which is vestigial in bacterial pentameric ligand-gated ion channels and was largely removed for crystallization of the Caenorhabditis elegans glutamate-activated pentameric ligand-gated ion channels. We created 5-HT3A subunit loop truncation mutants, in which sequences framing the putative portals were retained, to determine the minimum number of residues required to maintain their functional integrity. Truncation to between 90 and 75 amino acids produced 5-HT3A receptors with unaltered rectification. Truncation to 70 residues abolished rectification and increased γ. These findings reveal a critical M3-M4 loop length required for functions attributable to cytoplasmic portals. Examination of all 44 subunits of the human neurotransmitter-activated Cys-loop receptors reveals that, despite considerable variability in their sequences and lengths, all M3-M4 loops exceed 70 residues, suggesting a fundamental requirement for portal integrity.

Keywords: Cys-loop Receptors; GABA Receptors; Glycine Receptors; Ion Channels; Nicotinic Acetylcholine Receptors; Serotonin; Structural Biology.

FIGURE 1.

The HA stretch is the determinant of 5-HT₃ receptor rectification. A, diagrams representing the 5-HT3A/B chimeras, which were made by substituting different regions of the 5-HT3A (white) subunit with that of the 5-HT3B (gray) subunit. Chimeras were expressed in HEK cells, and currents were recorded at potentials between −60 and +60 mV in 20-mV increments. B, graph of the relationship between holding potential (V) in mV and current amplitude expressed relative to that of the current recorded at −60 mV. A near linear current-voltage relationship was exhibited by receptors that contained the HA stretch of the 5-HT3B subunit within the M3-M4 loop (5-HT3B and C3 and C4 constructs). As reported previously, the 5-HT3B subunit, C1, C2, and C3 required co-expression with the 5-HT3A subunit for function (5, 7).

FIGURE 2.

The HA stretch contains the determinants of inward rectification in 5-HT₃A receptors. A, ribbon diagram (left panel) based on the T. marmorata structure and alignment of human, mouse, and rat 5-HT3A and 5-HT3B subunit HA stretch residues (right panel). The three conserved arginine residues (432 at HA −4′, 436 at HA 0′, and 440 at HA 4′) in the human 5-HT3A subunit previously shown to affect single channel conductance are highlighted, along with the corresponding residues in the 5-HT3B subunit (13). These HA stretch Arg residues frame the cytoplasmic portals in a 5-HT₃A receptor homology model (13) based on the T. marmorata nACh receptor structure (9). Also highlighted in the alignment is Arg-426 (at −10′). B, systematic replacement of arginines within the 5-HT3A subunit HA stretch by corresponding residues in the 5-HT3B subunit reveals that the HA stretch contains the determinants for rectification. Data are represented as the ratio of 5-HT-evoked currents at +60 mV and −60 mV (I_{+60mV/−60mV}) in WT 5-HT₃A and 5-HT₃AB (black bars) and mutant 5-HT₃A receptors (open bars for single mutants and gray bars for double mutants). 5-HT₃A receptors display inward rectification as revealed by the below unitary I_{+60mV/−60mV}. 5-HT₃AB receptors on the other hand display a linear I-V relationship. Amino acid substitutions in the 426, 432, and 440 positions alone, or in combination, did not alter I_{+60mV/−60mV}. However, R436D produced a significant increase in I_{+60mV/−60mV} (p < 0.05, one-way ANOVA). R436D in combination with R432Q and/or R440A also produced receptors with a unitary I_{+60mV/−60mV} (*, p < 0.05, one-way ANOVA). Taken together, these data demonstrate that residues within the HA stretch govern I-V relationship properties of 5-HT₃A receptors.

FIGURE 3.

The charge and volume of residue 436 are determinants of inward rectification. Graphical representations of the relationship between I_{+60mV/−60mV} and either the amino acid charge or the volume of the HA 0′ residue are shown. Arg-436 was replaced by amino acids with differing properties. A, graph of the relationship between the change in charge, caused by the substitution of Arg-436, and I_{+60mV/−60mV}. Replacement of the positively charged Arg by an uncharged residue corresponds to a change of charge of 1, whereas its replacement by a negatively charged residue corresponds to a change of charge of 2. There was a significant correlation between the change in charge at position 436 and the rectification index (Pearson coefficient = 0.61; p = 0.006). The straight line indicates the linear fit through the data (R² = 0.37). B, relationship between the change in amino acid charge at position 436 restricted to charged and polar residues. Exclusion of the nonpolar residues improved the correlation coefficient (Pearson coefficient = 0.83; p = 0.004) and the linear fit (R² = 0.68). C, graph of the relationship between the volume of the amino acid at position 436 and I_{+60mV/−60mV}. In general, larger amino acids at position 436 were associated with more inward rectification. Indeed, there was a significant correlation between the volume of the residue at position 436 and the rectification index (Pearson coefficient = −0.62; p = 0.005). This relationship was fitted by a linear regression with an R² value of 0.38. D, exclusion of nonpolar residues also improved the correlation between the volume and the rectification index (Pearson coefficient = −0.72; p = 0.02) and the linear fit (R² = 0.51). Amino acid volumes were determined using Spartan ′04 software (see “Materials and Methods”).

FIGURE 4.

M3-M4 loop truncation alters the properties of 5-HT₃A receptors. A, alignment of wild-type 5-HT3A subunit M3-M4 region with truncation constructs. Truncation mutants are named according to the number of residues removed from the wild-type sequence. Truncation of 10 amino acids close to M3 (L-10(1) and L-10(2)) produced nonfunctional receptors (see “Results”). B, representative whole cell currents evoked by application of 100 μm 5-HT to HEK-293 cells expressing 5-HT₃A receptors (gray) or homomeric receptors composed of truncated (L-24, L-34, L-39, and L-44) constructs (black). 5-HT did not activate currents in cells expressing L-55 alone (see “Results”). C, a representative 5-HT-evoked current (black) recorded from a cell expressing the L-55 construct expressed in combination with 5-HT3A subunits. Currents are normalized to the amplitude of the exemplar current mediated by wild-type 5-HT₃A receptors to aid comparison of desensitization kinetics. D, the bar graphs show the fast and slow desensitization τ values (left panels) and the relative contributions of their amplitudes (right panels) for wild-type and truncated 5-HT₃A receptors. The sum of two exponentials was fitted to the decaying phase of the 5-HT-evoked current from the peak until the end of 5-HT application (see “Materials and Methods”). The use of a one-way ANOVA with post hoc Tukey's test revealed a significant difference in the τ values and their relative amplitudes for currents mediated by the L-39 receptors when compared with the other receptors tested (*, p < 0.05). E, the weighted τ for desensitization (τ_W) of 5-HT-evoked currents mediated by L-39 was slower than the equivalent values for the all the other homomeric receptors tested (*, p < 0.05, one-way ANOVA with post hoc Tukey's test).

FIGURE 5.

Evidence that the slow desensitization kinetics of L-39 is caused by the introduction of a phosphorylation consensus sequence. A, sequences of the full-length and truncated M3-M4 loops with putative phosphorylation sites highlighted. Sequences were analyzed using the Group-based Prediction (GPS 2.0) algorithm (15). Several phosphorylation consensus sequences were identified (shown in bold font). A putative Ca²⁺/calmodulin-dependent protein kinase site was identified in the L-39 construct at Thr-369 that was not found in the wild-type sequence or any of the other truncation constructs. B, the left panel contains traces of exemplar 5-HT (100 μm)-evoked currents mediated by L-34 receptors (gray trace) and L-34 (T369E) receptors in which Thr-369 was replaced by Glu, a phosphomimetic amino acid (black trace). The right panel contains traces of exemplar currents mediated by L-39 receptors (gray trace) and L-39 (T369A) receptors in which Thr-369 was substituted by Ala, thereby abolishing phosphorylation at this position. C, graph of the weighted τ values (τ_W) for 5-HT-evoked current desensitization. L-34 (T369E) receptors (black bar) exhibited slower desensitization kinetics (*, p < 0.05, Student's t test) when compared with the nonmutant L-34 receptors (gray bar). L-39 (T369A) receptors (black bar) desensitized faster than did nonmutated L-39 receptors (gray bar).

FIGURE 6.

The L-44 M3-M4 loop truncation abolishes inward rectification and increases γ. A, diagrams representing a predicted portal framed by adjacent HA stretches (top panel) and a scenario in which the portal has been disrupted due to short cytoplasmic loops connecting M3 to the bottom of the HA stretch (bottom panel). B, representative traces of 5-HT (100 μm)-evoked currents mediated by wild-type 5-HT₃A receptors and receptors formed by truncation constructs, recorded at +60 mV and −60 mV. 5-HT was applied for 2 s. The graph shows the relationship between M3-M4 loop length and the rectification indexes (I_{+60mV/−60mV}). Wild-type 5-HT₃A, L-24, L-34, and L-39 receptors all displayed inward rectification, as demonstrated by rectification indexes of ≤0.5. Larger truncations (L-44 and L-55), however, resulted in receptors that displayed more similar current amplitudes at +60 mV and −60 mV. The L-55 construct was expressed with the wild-type 5-HT3A subunit. The rectification indexes of currents mediated by L-44 and 5-HT3A + L-55 were significantly higher than those mediated by wild-type 5-HT3A receptors expressed alone (*, p < 0.05; one-way ANOVA). C, representative single channel recordings of L-44 at different holding potentials. 5-HT (10 μm) was pressure-applied to excised outside-out patches to evoke microscopic currents. Channel closed and open levels are indicated by gray dashed lines. Also shown are the all points amplitude histograms of the exemplar data with the corresponding Gaussian fits to indicate the mean closed and open levels. The plot of single channel current mediated by L-44 receptors versus voltage was fitted by a linear regression yielding a mean chord conductance of 16.3 ± 1.3 pS (n = 7).

FIGURE 7.

M3-M4 loop length of neurotransmitter-gated pentameric ion channels. The large cytoplasmic loop length of the human 5-HT3A subunit was established from the homology model based on the T. marmorata structure (13). The M3-M4 loop lengths of the other human Cys-loop receptor subunits were then estimated by aligning their sequences with that of the 5-HT3A subunit. A, graph of the loop lengths for cationic Cys-loop receptor subunits. B, graph of the loop lengths of anionic Cys-loop receptor subunits. Included for comparison are the loop lengths of the 5-HT3A L-39, which supported portal-mediated function, and L-44, which did not. Interestingly all of the neurotransmitter-gated pentameric ion channel subunits exceed the loop length of the L-44 construct (70 amino acids, indicated by the dotted line).