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Impact of intracellular domain flexibility upon properties of activated human 5-HT3 receptors.
Kozuska JL
,
Paulsen IM
,
Belfield WJ
,
Martin IL
,
Cole DJ
,
Holt A
,
Dunn SM
.
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It has been proposed that arginine residues lining the intracellular portals of the homomeric 5-HT3 A receptor cause electrostatic repulsion of cation flow, accounting for a single-channel conductance substantially lower than that of the 5-HT3 AB heteromer. However, comparison of receptor homology models for wild-type pentamers suggests that salt bridges in the intracellular domain of the homomer may impart structural rigidity, and we hypothesized that this rigidity could account for the low conductance. Mutations were introduced into the portal region of the human 5-HT3 A homopentamer, such that putative salt bridges were broken by neutralizing anionic partners. Single-channel and whole cell currents were measured in transfected tsA201 cells and in Xenopus oocytes respectively. Computational simulations of protein flexibility facilitated comparison of wild-type and mutant receptors. Single-channel conductance was increased substantially, often to wild-type heteromeric receptor values, in most 5-HT3 A mutants. Conversely, introduction of arginine residues to the portal region of the heteromer, conjecturally creating salt bridges, decreased conductance. Gating kinetics varied significantly between different mutant receptors. EC50 values for whole-cell responses to 5-HT remained largely unchanged, but Hill coefficients for responses to 5-HT were usually significantly smaller in mutants. Computational simulations suggested increased flexibility throughout the protein structure as a consequence of mutations in the intracellular domain. These data support a role for intracellular salt bridges in maintaining the quaternary structure of the 5-HT3 receptor and suggest a role for the intracellular domain in allosteric modulation of cooperativity and agonist efficacy.
Figure 1. Model of the 5-HT3A receptor obtained using the cryo-EM structure of the nACh receptor (PDB 2bg9; Unwin, 2005) as a template. This model was constructed using the conformation of the δ subunit as it exists at the interface with the α subunit and rotated around the channel axis to generate a homomeric channel with fivefold symmetry. (A) Two subunits of the receptor are shown as viewed from a position parallel to the membrane. The subunit on the right is shown in blue, the other subunit is coloured to illustrate distinct regions of the protein; ECD (cyan), TMD (violet) and ICD. The ICD consists of the TM1-TM2 loop (red), the beginning of the TM3-TM4 domain (green) and the MA helix (orange). (B) The MA helix as viewed looking down the ion pore from above the membrane. Other domains were deleted for clarity. Basic amino acid residues (blue) and acidic amino acid residues (red) project into the portal region between subunits. (C) The MA helix of two adjacent subunits as viewed from a position parallel to the membrane. The arginine residues previously identified as important determinants of single-channel conductance (Kelley et al., 2003) are highlighted in blue as well as K431, and the glutamate and aspartate residues that were investigated in this study are in red. Other charged residues not investigated are highlighted in cyan (arginine) and violet (aspartate) to illustrate the extent of charged residue presence in this region. A space-filling representation of these portals is shown in Supporting Information Figure S1. (D) A partial sequence alignment of the TM1-TM2 loop and MA helices of the 3A and 3B subunits.
Figure 2. The effect of MA helix mutations on the single-channel conductance of 5-HT3A receptors expressed in tsA201 cells. Receptors were expressed in tsA201 cells and outside-out patches were excised from these cells. The holding potential was −60 mV for all receptor constructs and 5-HT (10 μM) was used to evoke channel activity in all the homomeric receptors. 5-HT 100 μM was used to evoke activity of wild-type heteromeric receptors and 5-HT3AB(T431K,Q434E) receptors while 1 mM 5-HT was used for 5-HT3AB(Q432R,Q434E) receptors, to reflect the difference in 5-HT affinity. (A) Examples of the single-channel currents. Openings are downward deflections. QDA, 5-HT3A(QDA) receptor; QQN, 5-HT3A(QQN) receptor; QN, 5-HT3A(E437Q+D441N) receptor; D441N, 5-HT3A(D441N) receptor; E437Q, 5-HT3A(E437Q) receptor; E434Q, 5-HT3A(E434Q) receptor; K431T, 5-HT3A(K431T) receptor; E430Q, 5-HT3A(E430Q) receptor; E421Q, 5-HT3A(E421Q) receptor; AB, 5-HT3AB receptor; QQ-RE, 5-HT3AB(Q432R,Q434E) receptor and TQ-KE, 5-HT3AB(T431K,Q434E) receptor. (B) A comparison of the point conductance of each 5-HT3 receptor. Data are reported as mean ± SEM from at least three independent experiments. An anova was done to determine statistical differences from the 5-HT3A(QDA) receptor (*P < 0.05, **P < 0.01), the 5-HT3A(QQN) receptor (#P < 0.05, ##P < 0.01) and the 5-HT3AB receptor (∧P < 0.05).
Figure 3. Flexibility in 5-HT3A, 5-HT3A(QQN) and 5-HT3A(E434Q) receptors. (A) One subunit of each 5-HT3A mutant receptor is coloured by root mean square fluctuation relative to wild type (red denotes increased and blue decreased flexibility). Large increases in flexibility are observed at the top of the MA helices, close to the mutation sites and at the base of the TM3 helix (up to 0.4 Å) (inset). Figures generated using Pymol (The PyMOL Molecular Graphics System, Version 1.3 Schrödinger, LLC). (B) Distribution of portal widths for the three receptors from constrained geometric simulation. Histograms are fitted with cubic splines. The QQN (red lines) and E434Q (blue lines) mutant receptor portals are, on average, wider and fluctuate in diameter more than the wild-type 5-HT3A receptor portals (black lines).
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