Detection of the activity of ion channels and pores by circular dichroism spectroscopy: G‐quartets as functional CD probes within chirogenic vesicles

Abstract

We introduce a method to detect the activity of ion channels and pores with circular dichroism (CD) spectroscopy. G-quartets obtained by potassium templated self-assembly of 5'-guanosine monophosphate (GMP) are used as CD probes, the bee-toxin melittin as representative pore and gramicidin A as representative ion channel. To detect the activity of pores with CD spectroscopy, vesicles were loaded with GMP at concentrations above the dissociation constant (K(D)) of G-quartets. GMP efflux through added pores was detectable as CD silencing due to G-quartet disassembly by local dilution. The Hill plot of melittin with Hill coefficient n = 4 was faithfully reproduced with CD detection. The same was true for counterion-activated cell-penetrating peptides, confirming their ability to mediate the export of anions such as GMP. The same method is not applicable to the CD detection of the activity of ion channels because GMP efflux does not occur. To do that, the potassium selectivity of G-quartets was used. Namely, vesicles were loaded with GMP at concentrations above the dissociation constant (K(D)) of G-quartets stabilized by potassium. External M/K cation exchange resulted in CD silencing by M/K antiport through the added ion channel followed by G-quartet disassembly within the vesicle. Reversal of the direction of M/K antiport was achieved with Cs-loaded vesicles and detected as the appearance of the CD signature of G-quartets in response to K influx. Corroborative examples with gramicidin A include CD detection of the Eisenman I selectivity sequence and the Hill coefficient.

An artificial primitive mimic of the Gramicidin-A channel

“…The single files of water acting as lubricant between the internal surface of T-channel and diffusing ions, imposes a net dipolar water orientation that influence the conduction of ions envisioned to diffuse along the dipolar hydrophilic wires. Such correlation between two simple dissymmetric properties, that is, the chiral inner pore superstructure [34][35][36] and oriented dipolar water wires, might contribute to conceive novel strategies for artificial ion diffusion processes. Conversely, the molecular-scale hydrodynamics of water and ionic species within such simple artificial channels could be of crucial relevance for other biological scenarios.…”

Chirality sensing with pores: Reactive signal amplifiers for otherwise undetectable small molecules

“…Studies on detectors beyond fluorescence probes include G-quartets as circular dichroism (CD) probes. 19 Here, we show that the amplifier concept expands the use of chirality sensing with pores toward otherwise undetectable small molecules.…”

Hydrazinoanthrylboronic acids as exciton‐coupled circular dichroism (ECCD) probes for multivalent catechols, particularly epigallocatechin gallate

“…In our ongoing program on CD methods development at the ''bio-nano'' interface, we have introduced ECCD probes to detect osmotic pressure 35 or the activity of ion channels and pores. 36 Many elegant biosensors and chemosensors that have appeared over the past decades, however, operate usually with colorimetric, fluorometric, or electrochemical detection. [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][37][38][39][40][41][42][43][44][45][46][47][48][49][50] This includes much inspiring work from the groups of Shinkai, James, Anslyn, Wang, Smith, Schrader, and others on the use of boronic acids to sense sugars, a-hydroxy acids and catechols.…”