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

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 beetoxin 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.