title: Inhibition of K/HCO(3) cotransport in squid axons by quaternary ammonium ions.
abstract: 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 (pH(i)). With an inwardly directed K/HCO(3) 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 pH(i) increase, as measured with pH-sensitive microelectrodes. We dialyzed axons to pH(i) 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% CO(2)/12 mm HCO(3)(-), which (i) caused membrane potential to become transiently very positive, and (ii) caused a rapid pHi decrease, due to CO(2) influx, followed by a slower plateau-phase pH(i) increase, due to inward cotransport of K(+) and HCO(3)(-). With no QA(+) in the DF, J(HCO3) was approximately 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 approximately 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 approximately 20 pmole cm(-2) sec(-1) for tetramethylammonium (TMA(+)), approximately 24 for TEA(+), approximately 10 for tetrapropylammonium (TPA(+)), and virtually zero for tetrabutylammonium (TBA(+)). The apparent K(i) value for TBA(+) is approximately 0.86 mm. The most potent inhibitor was phenyl-propyltetraethylammonium (PPTEA(+)), with an apparent K(i) of approximately 91 microm. Thus, trans-side quaternary ammonium ions inhibit K/HCO(3) influx in the potency sequence PPTEA(+) > TBA(+) > TPA(+) > TEA(+) congruent with TMA(+). The identification of inhibitors of the K/HCO(3) cotransporter, for which no inhibitors previously existed, will facilitate the study of this transporter.
authors.affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA. bruce.davis@yale.edu
authors.forename: B A
authors.initials: BA
authors.lastname: Davis
authors.forename: E M
authors.initials: EM
authors.lastname: Hogan
authors.forename: G J
authors.initials: GJ
authors.lastname: Cooper
authors.forename: E
authors.initials: E
authors.lastname: Bashi
authors.forename: J
authors.initials: J
authors.lastname: Zhao
authors.forename: W F
authors.initials: WF
authors.lastname: Boron
mesh_terms: D000818:Animals; D001369:Axons / Q000187:drug effects* / Q000378:metabolism; D001639:Bicarbonates / Q000378:metabolism / Q000494:pharmacology; D049832:Decapodiformes; D004305:Dose-Response Relationship, Drug; D005731:Ganglionic Stimulants / Q000494:pharmacology; D007477:Ions; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D026902:Potassium Channel Blockers / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D027982:Sodium-Bicarbonate Symporters / Q000037:antagonists & inhibitors*; D019789:Tetraethylammonium / Q000494:pharmacology
chemical_list: D001639:Bicarbonates; D005731:Ganglionic Stimulants; D007477:Ions; D026902:Potassium Channel Blockers; D000644:Quaternary Ammonium Compounds; D027982:Sodium-Bicarbonate Symporters; C052377:tetrapropylammonium; D019789:Tetraethylammonium; C009405:tetrabutylammonium; C027917:tetramethylammonium; D011188:Potassium