Inhibition of K/HCO(3) cotransport in squid axons by quaternary ammonium ions.

Previous squid-axon studies identified a novel K/HCO3 cotransporter that is insensitive to disulfonic stilbene derivatives. This cotransporter presumably responds to intracellular alkali loads by moving K+ and HCO− 3 out of the cell, tending to lower intracellular pH (pHi). With an inwardly directed K/HCO3 gradient, the cotransporter mediates a net uptake of alkali (i.e., K+ and HCO− 3 influx). Here we test the hypothesis that intracellular quaternary ammonium ions (QA+) inhibit the inwardly directed cotransporter by interacting at the intracellular K+ site. We computed the equivalent HCO− 3 influx (J HCO3) mediated by the cotransporter from the rate of pHi increase, as measured with pH-sensitive microelectrodes. We dialyzed axons to pHi 8.0, using a dialysis fluid (DF) free of K+, Na+ and Cl−. Our standard artificial seawater (ASW) also lacked Na+, K+ and Cl−. After halting dialysis, we introduced an ASW containing 437 mm K+ and 0.5% CO2/12 mm HCO− 3, which (i) caused membrane potential to become transiently very positive, and (ii) caused a rapid pHi decrease, due to CO2 influx, followed by a slower plateau-phase pHi increase, due to inward cotransport of K+ and HCO− 3. With no QA+ in the DF, J HCO3 was ∼58 pmole cm−2 sec−1. With 400 mm tetraethylammonium (TEA+) in the DF, J HCO3 was virtually zero. The apparent K i for intracellular TEA+ was ∼78 mm, more than two orders of magnitude greater than that obtained by others for inhibition of K+ channels. Introducing 100 mm inhibitor into the DF reduced J HCO3 to ∼20 pmole cm−2 sec−1 for tetramethylammonium (TMA+), ∼24 for TEA+, ∼10 for tetrapropylammonium (TPA+), and virtually zero for tetrabutylammonium (TBA+). The apparent K i value for TBA+ is ∼0.86 mm. The most potent inhibitor was phenyl-propyltetraethylammonium (PPTEA+), with an apparent K i of ∼91 μm. Thus, trans-side quaternary ammonium ions inhibit K/HCO3 influx in the potency sequence PPTEA+ > TBA+ > TPA+ > TEA+≅ TMA+. The identification of inhibitors of the K/HCO3 cotransporter, for which no inhibitors previously existed, will facilitate the study of this transporter.