Chemical and genetic engineering of selective ion channel-ligand interactions

Abstract

Ionic flux mediates essential physiological and behavioral functions in defined cell populations. Cell type-specific activators of diverse ionic conductances are needed for probing these effects. We combined chemistry and protein engineering to enable the systematic creation of a toolbox of ligand-gated ion channels (LGICs) with orthogonal pharmacologic selectivity and divergent functional properties. The LGICs and their small-molecule effectors were able to activate a range of ionic conductances in genetically specified cell types. LGICs constructed for neuronal perturbation could be used to selectively manipulate neuron activity in mammalian brains in vivo. The diversity of ion channel tools accessible from this approach will be useful for examining the relationship between neuronal activity and animal behavior, as well as for cell biological and physiological applications requiring chemical control of ion conductance.

Fig. 1

“Bump-hole” approach to engineer selective ion channel-ligand interactions. (A) LGICs composed of ligand binding domain (LBD) and ion pore domain (IPD) modules. LBD mutations yield a Pharmacologically Selective Actuator Module (PSAM) that selectively binds Pharmacologically Selective Effector Molecules (PSEMs, red) but not the endogenous ligand (acetylcholine, ACh, yellow). PSEMs do not bind the unmodified LBD. (B) Chemical structures of ACh, nicotine, and PNU-282987. (C) Homology model of the α7 nAChR LBD with a docked agonist at the interface between two protomers (purple and green). Residues W77, Q79, Q139, and L141 were targeted for mutagenesis. (D) Current evoked by PNU-282987 application to an HEK 293 cell expressing α7-5HT3. A large peak current (I_peak) rapidly decays to a persistent, steady state current (I_ss). Black line indicates time course of ligand application. Scale bars: 200 pA, 0.5 s. Inset, Dose response curves (normalized to maximum response) from MP assay correspond to I_ss (n = 3). (E) Color map showing activity and cell surface expression of mutated α7-5HT3 channels. Responses to ACh (100 μM), nicotine (100 μM), PNU-282987 (10 μM), and binding of α-Bungarotoxin (Bgt)-Alexa594 were normalized to the response of the unmodified channel to ACh (100 μM) or α-Bgt-Alexa594 binding. Error bars are s.e.m.

Fig. 2

Selective interactions between ligands and mutated α7-5HT3 chimeric ion channels. (A) Color map showing EC50s for mutated α7-5HT3 with ACh, nicotine, and aminoquinuclidine benzamides. In order to highlight molecules that are highly selective for mutated α7-5HT3 chimeric receptors, only the activity of molecules with EC50 > 30 μM against unmodified α7-5HT3 (top row) are shown for the mutated receptors. (B) Dose response curves for compounds against mutated channels and showing negligible activation of unmodified α7-5HT3. EC50_MP in parentheses. Normalization is to the maximum response to each compound or to ACh (for unmodified α7-5HT3). (C) Dose response curves for mutated ion channel-ligand interactions that show orthogonal pharmacology. Normalization is to maximum response for each compound across mutated channels. (D) Chemical structures of ligands in B and C. Error bars are s.e.m.

Fig. 3

PSAM/IPD chimeric ion channels for neuron activation and silencing. (A) Confocal projection image of α-Bgt-Alexa594 labeling in a cortical brain slice expressing PSAM^Q79G,Q139G-5HT3 HC receptors. Left, Laminar boundaries. (B) Depolarization of layer 2/3 cortical neurons (in tetrodotoxin and CNQX) via PSAM-5HT3 HC channels by PSEMs applied at 10 μM. PSEM application (30 μM) to control neurons electroporated with GFP-expressing vector (green) did not depolarize cells. Sample sizes in parentheses. (C) Cell attached recordings of PSAM-5HT3 HC channel activation of layer 2/3 cortical neurons. Scale bar: 50 pA. Ligand application, 120 s. (D) Ligand selectivity: layer 2/3 cortical neurons expressing PSAM^L141F,Y115F-5HT3 HC are activated by PSEM^89S but not PSEM^22S. (E) Top, Confocal projection image of α-Bgt labeling in a cortical brain slice expressing PSAM^L141F,Y115F-GlyR receptors. Bottom, dendritic segment. (F,G) PSAM^L141F,Y115F-GlyR and PSEM^89S (10 μM, black, n = 16), but not PSEM^89S alone (30 μM, white, n = 10), reversibly reduce cellular input resistance, R_in. Shown in (G) is R_in normalized to pre-PSEM^89S application (PRE); values are displayed for PSEM^89S application and after 3 min wash-out (WASH). (H) PSAM^L141F,Y115F-GlyR reversibly reduces excitability of cortical neurons to depolarizing current injection in the presence of PSEM^89S. (I) Rheobase, normalized to pre-PSEM^89S application. Error bars are s.e.m.

Fig. 4

Stringent neuronal silencing test of PSAM^L141F,Y115F-GlyR for suppressing AGRP neuron-evoked feeding behavior. (A) Construct for a Cre-dependent recombinant adeno-associated viral vector with an inverted bicistronic open reading frame for PSAM^L141F,Y115F-GlyR and ChR2 under control of a FLEX-switch [two antiparallel pairs of heterotypic loxP sites (triangles)]. (B) Diagram illustrating injection site (left) and confocal images (right) taken from brain slices of virally transduced Agrp-cre mice showing co-expression of PSAM^L141F,Y115F-GlyR (green, α-Bgt-Alexa488) and ChR2 (red, anti-HA). (C) Cell-attached recording showing suppression of light-evoked bursts of action potential currents (10 Hz light pulses for 1 s, represented by blue dots) following application and then wash-out of PSEM^89S (above). Bursts are expanded for selected time points. Below is spike success rate (percent) before, during and after PSEM^89S application. (D) Successful light-evoked spikes per burst (n = 8 cells). (E) Food intake on successive days resulting from photostimulation in an Agrp-cre mouse co-expressing ChR2 and PSAM^L141F,Y115F-GlyR after saline (Baseline and Recovery) or PSEM^89S [intraperitoneal injection (inj.), marked with red arrow]. (F) Evoked food intake normalized to initial photostimulation session. Injection of PSEM^89S reduced photostimulation-evoked eating in Agrp-cre mice co-expressing ChR2 and PSAM^L141F,Y115F-GlyR (30 mg/kg, blue diamond, n = 5 mice) but not in Agrp-cre mice expressing only ChR2 (50 mg/kg, black circle, n = 6 mice). Error bars are s.e.m.