Using_VVG small_JJ molecules_NN2 to_TO facilitate_VVI exchange_NN1 of_IO bicarbonate_NN1 and_CC chloride_NN1 anions_NN2 across_II liposomal_JJ membranes_NN2 Bicarbonate_NN1 is_VBZ involved_JJ in_II a_AT1 wide_JJ range_NN1 of_IO biological_JJ processes_NN2 ,_, which_DDQ include_VV0 respiration_NN1 ,_, regulation_NN1 of_IO intracellular_JJ pH_NN1 and_CC fertilization_NN1 ._. 
In_II this_DD1 study_NN1 we_PPIS2 use_VV0 a_AT1 combination_NN1 of_IO NMR_NP1 spectroscopy_NN1 and_CC ion-selective_JJ electrode_NN1 techniques_NN2 to_TO show_VVI that_CST the_AT natural_JJ product_NN1 prodigiosin_NN1 ,_, a_AT1 tripyrrolic_JJ molecule_NN1 produced_VVN by_II microorganisms_NN2 such_II21 as_II22 Streptomyces_NP2 and_CC Serratia_NP1 ,_, facilitates_VVZ chloride/bicarbonate_NN1 exchange_NN1 (_( antiport_NN1 )_) across_II liposomal_JJ membranes_NN2 ._. 
Higher_JJR concentrations_NN2 of_IO simple_JJ synthetic_JJ molecules_NN2 based_VVN on_II a_AT1 core_NN1 are_VBR also_RR shown_VVN to_TO facilitate_VVI this_DD1 antiport_NN1 process_NN1 ._. 
Although_CS it_PPH1 is_VBZ well_RR known_VVN that_CST proteins_NN2 regulate_VV0 Cl/HCO_NP1 exchange_NN1 in_II cells_NN2 ,_, these_DD2 results_NN2 suggest_VV0 that_CST small_JJ molecules_NN2 may_VM also_RR be_VBI able_JK to_TO regulate_VVI the_AT concentration_NN1 of_IO these_DD2 anions_NN2 in_II biological_JJ systems_NN2 ._. 
Bicarbonate_NN1 is_VBZ an_AT1 important_JJ anion_NN1 ._. 
It_PPH1 is_VBZ a_AT1 substrate_NN1 in_II photosynthesis_NN1 ,_, it_PPH1 regulates_VVZ intra-_JJ and_CC extracellular_JJ pH_NN1 (_( ref._NN1 2_MC )_) ,_, it_PPH1 is_VBZ generated_VVN during_II cellular_JJ respiration_NN1 from_II carbon_NN1 dioxide_NN1 and_CC it_PPH1 acts_VVZ as_II a_AT1 cellular_JJ signal_NN1 to_TO activate_VVI sperm_NN for_IF fertilization4_FO ._. 
Under_II physiological_JJ conditions_NN2 most_RRT dissolved_VVN inorganic_JJ carbon_NN1 exists_VVZ as_CSA bicarbonate_NN1 ._. 
Bicarbonate_NN1 can_VM not_XX diffuse_VVI freely_RR across_II lipid_JJ membranes_NN2 ,_, and_CC bicarbonate_NN1 transport_NN1 is_VBZ facilitated_VVN in_RR21 vivo_RR22 by_II membrane-bound_JJ proteins_NN2 that_CST function_NN1 by_II31 means_II32 of_II33 Cl_FO /HCO_NN1 exchange_NN1 or_CC Na/HCO_FU co-transport_NN1 mechanisms_NN2 ._. 
Dysregulation_NN1 of_IO bicarbonate_NN1 transport_NN1 can_VM lead_VVI to_II conditions_NN2 such_II21 as_II22 cystic_JJ fibrosis_NN1 ,_, heart_NN1 disease_NN1 and_CC infertility_NN1 ._. 
The_AT lack_NN1 of_IO structural_JJ data_NN for_IF these_DD2 proteins_NN2 means_VVZ that_CST little_DA1 is_VBZ known_VVN about_II the_AT anion-binding_JJ sites_NN2 that_CST modulate_VV0 their_APPGE affinity_NN1 and_CC selectivity_NN1 ._. 
Despite_II the_AT importance_NN1 of_IO transmembrane_NN1 bicarbonate_NN1 transport_NN1 ,_, no_AT published_JJ studies_NN2 have_VH0 examined_VVN the_AT use_NN1 of_IO "_" small_JJ "_" molecules_NN2 to_TO promote_VVI the_AT efficient_JJ transport_NN1 of_IO bicarbonate_NN1 anions_NN2 across_II lipid_JJ membranes_NN2 (_( in_II contrast_NN1 to_II the_AT growing_JJ body_NN1 of_IO work_NN1 on_II transmembrane_NN1 chloride_NN1 transport_NN1 )_) ._. 
In_II a_AT1 study_NN1 that_CST focused_VVD on_II chloride_NN1 transport_NN1 ,_, a_AT1 synthetic_JJ steroid-based_JJ receptor_NN1 was_VBDZ reported_VVN to_TO support_VVI a_AT1 detectable_JJ chloride_NN1 efflux_NN1 from_II liposomes_NN2 on_II the_AT addition_NN1 of_IO extravesicular_JJ bicarbonate_NN1 ._. 
The_AT challenge_NN1 of_IO achieving_VVG bicarbonate_NN1 transport_NN1 was_VBDZ eloquently_RR expressed_VVN by_II A._NP1 P._NP1 Davis_NP1 et_RA21 al._RA22 :_: A_ZZ1 specific_JJ goal_NN1 would_VM be_VBI a_AT1 mimic_NN1 of_IO Cl/HCO_NP1 exchangers_VVZ that_CST play_VV0 important_JJ roles_NN2 in_II red_JJ blood_NN1 cells_NN2 and_CC epithelial_JJ tissues_NN2 ._. 
The_AT design_NN1 challenge_VV0 here_RL is_VBZ to_TO produce_VVI a_AT1 transporter_NN1 that_CST can_VM extract_VVI the_AT very_RG hydrophilic_JJ bicarbonate_NN1 anion_NN1 into_II the_AT lipophilic_JJ interior_NN1 of_IO a_AT1 bilayer_NN1 membrane_NN1 ._. 
Prodigiosin_NP1 (_( 1_MC1 )_) is_VBZ a_AT1 natural_JJ product_NN1 produced_VVN by_II microorganisms_NN2 such_II21 as_II22 Streptomyces_NP2 and_CC Serratia_NP1 ._. 
This_DD1 tripyrrolic_JJ metabolite_NN1 has_VHZ potent_JJ inmunosuppressive_JJ and_CC anticancer_NN1 activities_NN2 ._. 
Prodigiosin_NN1 causes_VVZ selective_JJ apoptosis_NN1 of_IO cancer_NN1 cells_NN2 ,_, and_CC its_APPGE structural_JJ analogue_JJ obatoclax_NN1 is_VBZ in_II clinical_JJ trials_NN2 for_IF the_AT treatment_NN1 of_IO cancer_NN1 ._. 
The_AT origin_NN1 of_IO prodigiosin_NN1 's_GE biological_JJ activity_NN1 has_VHZ yet_RR to_TO be_VBI established_VVN unambiguously_RR ,_, although_CS it_PPH1 facilitates_VVZ co-transport_NN1 of_IO HCl_NP1 and_CC anion_NN1 exchange_NN1 of_IO chloride_NN1 across_II lipid_JJ bilayers_NN2 ._. 
Indeed_RR ,_, the_AT anticancer_NN1 activity_NN1 of_IO prodigiosin-like_JJ compounds_NN2 has_VHZ been_VBN related_VVN to_II their_APPGE activity_NN1 as_CSA transmembrane_NN1 chloride_NN1 carriers_NN2 ._. 
The_AT presence_NN1 of_IO hydrogen-bond_JJ donors_NN2 and_CC acceptors_NN2 in_II prodigiosin_NN1 1_MC1 suggested_VVD that_CST it_PPH1 might_VM function_VVI as_II a_AT1 receptor_NN1 and_CC an_AT1 agent_NN1 for_IF the_AT membrane_NN1 transport_NN1 of_IO bicarbonate_NN1 (_( Fig._NN1 1_MC1 )_) ._. 
We_PPIS2 also_RR investigated_VVD the_AT bicarbonate_NN1 transport_NN1 ability_NN1 of_IO 2-4_MCMC to_TO ascertain_VVI whether_CSW these_DD2 compounds_NN2 function_VV0 as_CSA Cl/HCO_NP1 anti-porters_NN2 ,_, especially_RR in_II comparison_NN1 to_II prodigiosin_NN1 1_MC1 ._. 
We_PPIS2 recently_RR reported_VVD the_AT transmembrane_NN1 chloride-transport_NN1 activity_NN1 of_IO 2_MC (_( ref._NN1 31_MC )_) ._. 
Isophthalamides_NN2 have_VH0 convergent_JJ amide_NN1 NH_NP1 groups_NN2 that_CST can_VM form_VVI hydrogen_NN1 bonds_NN2 with_IW anions_NN2 ._. 
In_II the_AT case_NN1 of_IO 2_MC ,_, conformational_JJ control_NN1 of_IO the_AT anion-binding_JJ cleft_NN1 by_II intramolecular_JJ hydrogen_NN1 bonds_NN2 between_II the_AT 4,6-dihydroxy_FO units_NN2 and_CC the_AT neighbouring_JJ amide_NN1 carbonyls_NN2 resulted_VVN in_II an_AT1 improved_JJ anion_NN1 affinity_NN1 and_CC in_II optimal_JJ activity_NN1 for_IF transmembrane_NN1 transport_NN1 of_IO chloride_NN1 anion_NN1 ._. 
In_II fact_NN1 ,_, an_AT1 analogue_NN1 of_IO 2_MC that_CST lacked_VVD the_AT OH_UH groups_NN2 was_VBDZ shown_VVN not_XX to_TO function_VVI as_II a_AT1 membrane_NN1 transport_NN1 agent_NN1 for_IF chloride_NN1 ._. 
For_IF this_DD1 study_NN1 ,_, we_PPIS2 synthesized_VVD the_AT related_JJ isophthalamides_NN2 3_MC and_CC 4_MC ,_, functionalized_VVD with_IW different_JJ alkyl_NN1 substituents_NN2 ,_, as_CSA we_PPIS2 reasoned_VVD that_DD1 membrane_NN1 activity_NN1 might_VM be_VBI attenuated_VVN by_II the_AT identity_NN1 of_IO the_AT lipophilic_JJ tails_NN2 attached_VVN to_II the_AT isophthalamide_NN1 (_( Fig._NN1 1_MC1 )_) ._. 
Phospholipid_JJ vesicles_NN2 have_VH0 been_VBN extensively_RR used_VVN as_CSA models_NN2 for_IF biological_JJ membranes_NN2 ._. 
Unilamellar_JJ vesicles_NN2 of_IO a_AT1 defined_JJ size_NN1 are_VBR easily_RR produced_VVN with_IW control_NN1 of_IO the_AT entrapped_JJ solution_NN1 ._. 
These_DD2 liposomes_NN2 can_VM be_VBI suspended_VVN in_II an_AT1 external_JJ medium_NN1 of_IO different_JJ composition_NN1 and_CC the_AT transporter-facilitated_JJ release_NN1 of_IO encapsulated_JJ substrates_NN2 or_CC the_AT influx_NN1 of_IO substances_NN2 from_II the_AT external_JJ milieu_NN1 to_II the_AT interior_NN1 of_IO the_AT vesicles_NN2 can_VM be_VBI monitored_VVN by_II fluorescence_NN1 ,_, NMR_NP1 or_CC ion-selective_JJ electrode_NN1 techniques_NN2 ._. 
We_PPIS2 report_VV0 the_AT first_MD demonstration_NN1 that_CST transmembrane_NN1 Cl2_FO /HCO3_JJ exchange_NN1 is_VBZ facilitated_VVN by_II prodigiosin_NN1 1_MC1 and_CC synthetic_JJ receptors_NN2 2-4_MCMC ,_, and_CC the_AT NMR_NP1 spectro-scopic_JJ methods_NN2 used_VMK to_TO monitor_VVI directly_RR the_AT transport_NN1 of_IO bicarbonate_NN1 into_II lipid_JJ vesicles_NN2 ._. 
These_DD2 NMR_NP1 spectroscopy_NN1 studies_NN2 ,_, in_II combination_NN1 with_IW the_AT use_NN1 of_IO chloride-selective_JJ electrodes_NN2 ,_, allow_VV0 the_AT flux_NN1 of_IO both_DB2 components_NN2 of_IO an_AT1 antiport_NN1 process_NN1 to_TO be_VBI monitored_VVN ._. 
We_PPIS2 hope_VV0 that_CST these_DD2 studies_NN2 will_VM set_VVI the_AT stage_NN1 for_IF further_JJR development_NN1 of_IO selective_JJ transporters_NN2 for_IF bicarbonate_NN1 and_CC perhaps_RR ,_, in_II the_AT longer_JJR term_NN1 ,_, lead_VV0 to_II new_JJ approaches_NN2 for_IF treating_VVG diseases_NN2 caused_VVN by_II defective_JJ bicarbonate_NN1 transport_NN1 ._. 
Results_NN2 Prodigiosin_NN1 and_CC its_APPGE analogues_NN2 bind_VV0 chloride_NN1 in_II the_AT solid_JJ state_NN1 and_CC in_II solution_NN1 ._. 
Furthermore_RR ,_, the_AT use_NN1 of_IO chloride-selective_JJ dyes_NN2 and_CC electrodes_NN2 has_VHZ revealed_VVN that_DD1 prodigiosin_NN1 1_MC1 transports_VVZ chloride_NN1 anions_NN2 across_II lipid_JJ membranes_NN2 ._. 
Although_CS it_PPH1 was_VBDZ reported_VVN 50_MC years_NNT2 ago_RA that_DD1 prodigiosin_NN1 reacts_VVZ with_IW carbonic_JJ acid_NN1 to_TO give_VVI a_AT1 protonated_JJ adduct_NN1 ,_, no_AT direct_JJ evidence_NN1 has_VHZ been_VBN presented_VVN that_DD1 prodigiosin_NN1 1_MC1 can_VM bind_VVI bicarbonate_NN1 ._. 
We_PPIS2 studied_VVD the_AT anion-complexation_JJ properties_NN2 of_IO prodigiosin_NN1 1_MC1 by_II 1H-NMR_JJ titration_NN1 methods_NN2 in_II CD2Cl2_FO ._. 
These_DD2 NMR_NP1 spectroscopy_NN1 studies_NN2 showed_VVD that_DD1 bicarbonate_NN1 binds_VVZ to_II 1_MC1 ,_, which_DDQ caused_VVD shifts_NN2 of_IO proton_NN1 resonances_NN2 in_II prodigiosin_NN1 when_CS tetraethylammonium_NN1 bicarbonate_NN1 was_VBDZ added_VVN ._. 
The_AT NMR_NP1 signals_NN2 in_II 1_MC1 most_RRT affected_VVN by_II bicarbonate_NN1 addition_NN1 were_VBDR the_AT H2_FO proton_NN1 on_II the_AT A_ZZ1 ring_NN1 and_CC the_AT methyl_NN1 group_NN1 on_II the_AT C_NP1 ring_NN1 (_( see_VV0 Supplementary_JJ Information_NN1 )_) ._. 
These_DD2 carbon-bound_JJ protons_NN2 are_VBR expected_VVN to_TO be_VBI influenced_VVN most_DAT by_II anion_NN1 binding_VVG ,_, as_CSA they_PPHS2 are_VBR closest_JJT to_II prodigiosin_NN1 's_GE putative_JJ anion-binding_JJ cleft_NN1 (_( Fig._NN1 1_MC1 )_) (_( the_AT pyrrole_NN1 NH_NP1 protons_NN2 are_VBR not_XX visible_JJ in_II the_AT 1H-NMR_JJ spectrum_NN1 of_IO prodigiosin_NN1 in_II the_AT free-base_NN1 form_NN1 in_II CD2Cl2_FO )_) ._. 
The_AT changes_NN2 in_II these_DD2 chemical_JJ shifts_NN2 when_RRQ tetraethylammonium_NN1 bicarbonate_NN1 was_VBDZ added_VVN were_VBDR greater_JJR than_CSN those_DD2 for_IF the_AT same_DA protons_NN2 in_II 1_MC1 on_II the_AT addition_NN1 of_IO tetrabutylammonium_NN1 chloride_NN1 or_CC nitrate_NN1 (_( Ka=7.8_FO and_CC 7.0_MC M_NNO ,_, respectively_RR )_) ,_, which_DDQ presumably_RR reflects_VVZ the_AT higher_JJR basicity_NN1 of_IO the_AT bicarbonate_NN1 anion_NN1 ._. 
We_PPIS2 could_VM not_XX calculate_VVI a_AT1 stability_NN1 constant_NN1 for_IF the_AT prodigiosin-bicarbonate_JJ complex_NN1 because_CS ,_, as_II31 well_II32 as_II33 changes_NN2 in_II chemical_JJ shifts_NN2 for_IF 1_MC1 ,_, a_AT1 second_MD set_NN1 of_IO NMR_NP1 peaks_NN2 emerged_VVD during_II the_AT bicarbonate_NN1 titration_NN1 ._. 
This_DD1 slow_JJ exchange_NN1 process_NN1 may_VM be_VBI caused_VVN by_II a_AT1 higher_JJR order_NN1 complex_JJ formation_NN1 with_IW bicarbonate_NN1 or_CC by_II a_AT1 bicarbonate-triggered_JJ interconversion_NN1 of_IO rotamers_NN2 ._. 
Experiments_NN2 were_VBDR repeated_VVN with_IW the_AT protonated_JJ form_NN1 of_IO prodigiosin_NN1 as_II the_AT methanesulfonate_JJ salt_NN1 ._. 
Under_II the_AT same_DA experimental_JJ conditions_NN2 ,_, the_AT addition_NN1 of_IO bicarbonate_NN1 resulted_VVN in_II deprotonation_NN1 of_IO 1_MC1 H_ZZ1 ,_, as_CSA shown_VVN by_II loss_NN1 of_IO the_AT pyrrole_NN1 NH_NP1 resonances_NN2 in_II the_AT 1H-NMR_JJ spectrum_NN1 (_( see_VV0 Supplementary_JJ Information_NN1 )_) ,_, and_CC the_AT addition_NN1 of_IO chloride_NN1 caused_VVD a_AT1 downfield_NN1 shift_NN1 of_IO the_AT NH_NP1 resonances_NN2 ,_, which_DDQ indicates_VVZ hydrogen-bond_JJ formation_NN1 to_II the_AT halide_NN1 anion_NN1 ._. 
At_II pH_NN1 7.2_MC both_RR protonated_VVD and_CC free-base_NN1 forms_NN2 of_IO prodigiosin_NN1 are_VBR present_JJ and_CC it_PPH1 may_VM be_VBI that_CST any_DD putative_JJ antiport_NN1 process_NN1 involves_VVZ both_RR forms_VVZ ._. 
Although_CS a_AT1 stability_NN1 constant_NN1 for_IF bicarbonate_NN1 complexation_NN1 was_VBDZ not_XX obtained_VVN ,_, these_DD2 NMR_NP1 titrations_NN2 demonstrate_VV0 that_CST the_AT free-base_NN1 form_NN1 of_IO prodigiosin_NN1 1_MC1 binds_VVZ bicarbonate_NN1 in_II solution_NN1 ._. 
Electrospray_VV0 mass_JJ spectrometry_NN1 in_II negative_JJ mode_NN1 on_II a_AT1 solution_NN1 of_IO prodigiosin_NN1 1_MC1 in_II acetonitrile_NN1 revealed_VVD both_RR chloride_NN1 and_CC bicarbonate_NN1 adducts_NN2 (_( see_VV0 Methods_NN2 )_) ,_, which_DDQ provides_VVZ more_DAR evidence_NN1 for_IF complex_JJ formation_NN1 between_II prodigiosin_NN1 1_MC1 and_CC bicarbonate_NN1 ._. 
We_PPIS2 next_MD compared_VVD the_AT transmembrane_NN1 anion-transport_NN1 activity_NN1 of_IO the_AT natural_JJ product_NN1 1_MC1 with_IW those_DD2 of_IO the_AT synthetic_JJ chloride_NN1 transporters_NN2 2-4_MCMC ._. 
The_AT transmembrane_NN1 anion-transport_NN1 abilities_NN2 of_IO 1-4_MCMC were_VBDR evaluated_VVN by_II monitoring_VVG chloride_NN1 efflux_NN1 from_II unilamellar_JJ (_( POPC_NP1 )_) vesicles_NN2 using_VVG a_AT1 chloride-selective_JJ electrode_NN1 ._. 
These_DD2 studies_NN2 were_VBDR conducted_VVN using_VVG nitrate_NN1 in_II the_AT extravesicular_JJ solution_NN1 ._. 
Nitrate_NN1 is_VBZ more_RGR hydrophobic_JJ than_CSN bicarbonate_NN1 and_CC is_VBZ frequently_RR used_VVN to_TO assess_VVI chloride-transport_JJ efficiency_NN1 ._. 
Liposomes_NP1 were_VBDR loaded_VVN with_IW a_AT1 sodium_NN1 chloride_NN1 solution_NN1 and_CC suspended_VVN in_II a_AT1 sodium_NN1 nitrate_NN1 solution_NN1 ._. 
The_AT transporters_NN2 1-4_MCMC ,_, dissolved_VVN in_II a_AT1 small_JJ amount_NN1 (_( 10_MC ml_NNU )_) of_IO dimethylsulfoxide_NN1 (_( DMSO_NP1 )_) ,_, were_VBDR added_VVN to_II the_AT extravesicular_JJ solution_NN1 ,_, and_CC chloride_NN1 efflux_NN1 was_VBDZ monitored_VVN for_IF 300_MC s_ZZ1 ._. 
At_II the_AT end_NN1 of_IO the_AT experiment_NN1 the_AT vesicles_NN2 were_VBDR lysed_VVN by_II detergent_NN1 and_CC the_AT final_JJ value_NN1 normalized_VVD to_II equal_JJ the_AT complete_JJ chloride_NN1 efflux_NN1 ._. 
Prodigiosin_NN1 1_MC1 proved_VVD to_TO be_VBI a_AT1 potent_JJ chloride_NN1 transporter_NN1 using_VVG this_DD1 assay_NN1 ._. 
A_ZZ1 0.005%_FO molar_JJ carrier-to-lipid_JJ concentration_NN1 of_IO prodigiosin_NN1 1_MC1 showed_VVD similar_JJ transport_NN1 activity_NN1 to_II those_DD2 of_IO the_AT 0.1%_FO molarcarrier-to-lipid_JJ concentration_NN1 for_IF synthetic_JJ compounds_NN2 2-4_MCMC (_( Fig._NN1 2_MC )_) ._. 
These_DD2 carrier_NN1 loadings_NN2 were_VBDR able_JK to_TO complete_VVI chloride_NN1 efflux_NN1 within_II 300_MC s_ZZ1 ;_; the_AT isopentyl-substituted_JJ isophthalamide_NN1 3_MC was_VBDZ the_AT most_RGT active_JJ synthetic_JJ transporter_NN1 under_II these_DD2 conditions_NN2 ._. 
In_II the_AT assay_NN1 shown_VVN in_II Fig._NN1 2a_FO ,_, the_AT anion-transport_JJ activity_NN1 can_VM occur_VVI either_RR by_II H_ZZ1 /Cl_FU or_CC Na+/Cl_NP1 co-transport_NN1 or_CC by_II Cl2_FO /NO3_JJ exchange_NN1 ._. 
To_TO distinguish_VVI between_II these_DD2 alternative_JJ mechanisms_NN2 ,_, we_PPIS2 carried_VVD out_RP the_AT chloride-selective_JJ electrode_NN1 transport_NN1 assay_NN1 while_CS varying_VVG the_AT anion_NN1 in_II the_AT external_JJ medium_NN1 ._. 
If_CS transport_NN1 occurs_VVZ by_II an_AT1 anion-exchange_JJ mechanism_NN1 ,_, changes_NN2 in_II the_AT external_JJ anion_NN1 should_VM impact_VVI the_AT transport_NN1 rate_NN1 ,_, whereas_CS a_AT1 H/Cl_NP1 or_CC Na/Cl_FO co-transport_NN1 mechanism_NN1 should_VM not_XX be_VBI affected_VVN by_II the_AT external_JJ anion_NN1 ._. 
As_CSA depicted_VVN in_II Fig._NN1 2b_FO ,_, the_AT transport_NN1 assay_NN1 was_VBDZ repeated_VVN by_II suspending_VVG the_AT chloride-loaded_JJ vesicles_NN2 in_II a_AT1 sulfate-containing_JJ external_JJ medium_NN1 ._. 
As_II the_AT sulfate_NN1 dianion_NN1 carries_VVZ a_AT1 higher_JJR charge_NN1 and_CC is_VBZ significantly_RR more_RGR hydrophilic_JJ than_CSN the_AT nitrate_NN1 ion_NN1 ,_, transport_NN1 activity_NN1 of_IO compounds_NN2 1-4_MCMC should_VM be_VBI reduced_VVN if_CS the_AT mechanism_NN1 is_VBZ one_MC1 of_IO anion-exchange_NN1 ._. 
Indeed_RR ,_, with_IW sulfate_NN1 as_II the_AT external_JJ anion_NN1 ,_, no_AT chloride_NN1 efflux_NN1 from_II the_AT liposomes_NN2 was_VBDZ detected_VVN on_II the_AT addition_NN1 of_IO 1-4_MCMC ,_, which_DDQ supports_VVZ a_AT1 chloride/nitrate_NN1 exchange_NN1 (_( antiport_NN1 )_) mechanism_NN1 for_IF mediating_VVG anion_NN1 transport_NN1 across_II the_AT vesicle_NN1 bilayer_NN1 ._. 
Although_CS both_RR nitrate_NN1 and_CC bicarbonate_NN1 have_VH0 similar_JJ sizes_NN2 and_CC shapes_NN2 ,_, bicarbonate_NN1 is_VBZ significantly_RR more_RGR hydrophilic_JJ than_CSN nitrate_NN1 and_CC ,_, as_CSA has_VHZ been_VBN stressed11_FO ,_, it_PPH1 is_VBZ more_RGR challenging_JJ to_TO transport_VVI bicarbonate_NN1 than_CSN nitrate_NN1 across_II a_AT1 lipid_JJ bilayer_NN1 ._. 
Prompted_VVN by_II the_AT ability_NN1 of_IO prodigiosin_NN1 1_MC1 to_TO bind_VVI bicarbonate_NN1 and_CC by_II the_AT Cl2_FO /NO3_VVI anion-exchange_JJ activity_NN1 shown_VVN by_II 1-4_MCMC ,_, we_PPIS2 designed_VVD an_AT1 experiment_NN1 to_TO determine_VVI whether_CSW these_DD2 compounds_NN2 could_VM facilitate_VVI transmembrane_NN1 Cl2_FO /HCO3_JJ exchange_NN1 ._. 
Chloride-loaded_JJ vesicles_NN2 were_VBDR suspended_VVN in_II a_AT1 sulfate-containing_JJ medium_NN1 ._. 
After_II 2_MC minutes_NNT2 ,_, a_AT1 solution_NN1 of_IO bicarbonate_NN1 was_VBDZ added_VVN and_CC chloride_NN1 efflux_NN1 was_VBDZ monitored_VVN for_IF a_AT1 further_JJR 5_MC minutes_NNT2 ._. 
At_II the_AT end_NN1 of_IO the_AT experiment_NN1 the_AT vesicles_NN2 were_VBDR lysed_VVN to_TO calibrate_VVI the_AT experimental_JJ data_NN to_II 100%_NNU chloride_NN1 release_NN1 ._. 
The_AT results_NN2 shown_VVN in_II Fig._NN1 3_MC confirmed_VVD that_CST negligible_JJ chloride_NN1 efflux_NN1 was_VBDZ detected_VVN with_IW sulfate_NN1 as_II the_AT external_JJ anion_NN1 ._. 
Addition_NN1 of_IO bicarbonate_NN1 to_II the_AT extravesicular_JJ solution_NN1 switched_VVD on_RP the_AT chloride_NN1 efflux_NN1 in_II the_AT presence_NN1 of_IO 1-4_MCMC ,_, which_DDQ indicates_VVZ that_CST these_DD2 compounds_NN2 enable_VV0 Cl2_FO /HCO3_JJ antiport_NN1 across_II liposomal_JJ membranes_NN2 ._. 
As_CSA was_VBDZ observed_VVN for_IF Cl2_FO /NO3_JJ exchange_NN1 ,_, prodigiosin_NN1 1_MC1 (_( at_II 0.04%_FO molar_JJ carrier_NN1 to_II lipid_JJ )_) was_VBDZ more_RGR efficient_JJ than_CSN synthetic_JJ carriers_NN2 2-4_MCMC (_( 1%_NNU molar_JJ carrier_NN1 to_II lipid_JJ )_) in_II catalyzing_VVG Cl2_FO /HCO3_JJ transmembrane_NN1 exchange_NN1 ._. 
Under_II the_AT assay_NN1 conditions_NN2 (_( Fig._NN1 3_MC )_) ,_, the_AT addition_NN1 of_IO bicarbonate_NN1 induced_VVD small_JJ changes_NN2 (_( 0.2_MC units_NN2 )_) in_II the_AT pH_NN1 of_IO the_AT external_JJ medium_NN1 ._. 
We_PPIS2 carried_VVD out_RP control_NN1 experiments_NN2 with_IW 1-4_MCMC to_TO check_VVI the_AT possibility_NN1 that_CST chloride_NN1 efflux_NN1 was_VBDZ driven_VVN by_II a_AT1 pH_NN1 gradient_NN1 ._. 
NaOH_NN1 added_VVN to_II the_AT external_JJ medium_NN1 resulted_VVN in_II no_AT significant_JJ chloride_NN1 efflux_NN1 ._. 
Furthermore_RR ,_, the_AT addition_NN1 of_IO bicarbonate_NN1 solutions_NN2 to_II a_AT1 suspension_NN1 of_IO vesicles_NN2 without_IW transporters_NN2 1-4_MCMC resulted_VVN in_II no_AT chloride_NN1 efflux_NN1 ._. 
The_AT experiments_NN2 depicted_VVN in_II Fig._NN1 3_MC provided_CS strong_JJ ,_, yet_RR indirect_JJ ,_, evidence_NN1 that_CST transporters_NN2 1-4_MCMC move_NN1 bicarbonate_NN1 across_II lipid_JJ membranes_NN2 ._. 
We_PPIS2 next_MD used_VVD 13_MC C-NMR_JJ spectroscopy_NN1 to_TO verify_VVI that_CST these_DD2 transporters_NN2 facilitate_VV0 transmembrane_NN1 HCO3_FO /Cl2_JJ exchange_NN1 ._. 
We_PPIS2 developed_VVD experiments_NN2 that_CST use_VV0 paramagnetic_JJ Mn2_FO to_TO bleach_VVI the_AT 13_MC C-NMR_JJ signal_NN1 for_IF extravesicular_JJ H_ZZ1 CO3_FO ,_, which_DDQ allows_VVZ the_AT discrimination_NN1 of_IO extravesicular_JJ and_CC intravesicular_JJ HCO3_FO ._. 
We_PPIS2 based_VVD these_DD2 paramagnetic_JJ NMR_NP1 protocols_NN2 on_II previous_JJ experiments_NN2 that_CST both_DB2 monitored_VVD transmembrane_NN1 chloride_NN1 transport_NN1 in_II liposomes_NN2 by_II Cl-NMR_JJ spectroscopy_NN1 (_( refs_NN2 39_MC ,_, 40_MC )_) and_CC showed_VVD that_CST intracellular_JJ and_CC extracellular_JJ H13_FO CO3_FO 2could_FO be_VBI distinguished_VVN in_II plant_NN1 cells_NN2 ._. 
Figure_NN1 4_MC shows_VVZ data_NN from_II the_AT first_MD set_NN1 of_IO NMR_NP1 spectroscopy_NN1 experiments_NN2 conducted_VVN to_TO illustrate_VVI transporter-mediated_JJ HCO3_FO 2_MC /Cl2_JJ exchange_NN1 ._. 
These_DD2 NMR_NP1 spectroscopy_NN1 experiments_NN2 were_VBDR done_VDN under_II similar_JJ conditions_NN2 to_II those_DD2 described_VVN for_IF the_AT chloride-selective_JJ electrode_NN1 experiments_NN2 in_II Fig._NN1 3_MC ._. 
Thus_RR ,_, egg_NN1 yolk_NN1 phosphatidylcholine_NN1 (_( EYPC_NP1 )_) liposomes_NN2 (_( 5_MC mm_NNU )_) filled_VVD with_IW 450_MC mM_NNU NaCl_NN1 were_VBDR suspended_VVN in_II a_AT1 sulfate_NN1 solution_NN1 and_CC 50_MC mM_NNU H13_FO CO3_FO 2_MC added_VVD to_II the_AT NMR_NP1 sample_NN1 ._. 
