Structural requirements in the transmembrane domain of GLIC revealed by incorporation of noncanonical histidine analogs

Abstract

The cyanobacterial pentameric ligand-gated ion channel GLIC, a homolog of the Cys-loop receptor superfamily, has provided useful structural and functional information about its eukaryotic counterparts. X-ray diffraction data and site-directed mutagenesis have previously implicated a transmembrane histidine residue (His234) as essential for channel function. Here, we investigated the role of His234 via synthesis and incorporation of histidine analogs and α-hydroxy acids using in vivo nonsense suppression. Receptors were expressed heterologously in Xenopus laevis oocytes, and whole-cell voltage-clamp electrophysiology was used to monitor channel activity. We show that an interhelix hydrogen bond involving His234 is important for stabilization of the open state, and that the shape and basicity of its side chain are highly sensitive to perturbations. In contrast, our data show that two other His residues are not involved in the acid-sensing mechanism.

Figure 1. Several Views of the GLIC Channel

(A) Crystal structure of GLIC in a presumed open state (PDB ID 3EHZ). Histidines studied here are highlighted. (B) Top-down overlay of a locally closed GLIC mutant structure (blue, PDB ID 3TLU) with the open channel structure (green), omitting the extracellular domain. Note the movement of the M2 helix associated with channel opening. (C) Side view overlay of locally closed (blue) and open (green) GLIC structures. The orientation of the depicted residues suggests the formation of a hydrogen bond (dashed line) between His234 and the backbone carbonyl of I258 upon channel activation.

Figure 2. Maximal Currents Observed for Mutants at His126

These data were obtained from the same batch of oocytes to minimize oocyte variability. Values are relative to wild-type (100%); data = mean ± SEM, n = 3–5.

Figure 3. Backbone Ester Mutagenesis

Incorporation of phenyllactic acid at Phe259 introduces a backbone ester mutation, converting a strong hydrogen bond (solid line) to a weaker hydrogen bond (dashed line).

Figure 4. Synthetic Schemes

Preparation of (top) 2-CH₃His and (bottom) 2-CF₃His for ligation to suppressor tRNA.

Figure 5. Incorporation of His Analogues into Functional Receptors

(A and B) Control experiments demonstrate robust nonsense suppression and membrane trafficking with histidine (dotted) and synthetic histidine anlaogs 2-CH₃His (solid) and 2-CF₃His (dashed) at His126 (A) and His276 (B). Mean normalized current responses are plotted from 12–20 oocytes per condition, with SE indicated. (C) Space-filling models of 2-CH₃-imidazole (top) and 2-CF₃-imidazole (bottom), showing they are comparable in size. See also Figure S2.

Figure 6. Studies of His Analogues at His234

(A) Space-filling model of His234 and surrounding residues in the presumed open state structure (PDB ID 3EHZ). (B) Whole-cell current responses of oocytes expressing GLIC mutants to buffer applications at pH 7.5, 6.5, 5.5, and 4.5. See also Figure S3.