K(+)- and HCO3(-)-dependent acid-base transport in squid giant axons. I. Base efflux.

Abstract

We used microelectrodes to monitor the recovery (i.e., decrease) of intracellular pH (pHi) after using internal dialysis to load squid giant axons with alkali to pHi values of 7.7, 8.0, or 8.3. The dialysis fluid (DF) contained 400 mM K + but was free of Na + and C1-. The artificial seawater (ASW) lacked Na +, K +, and CI-, thereby eliminating effects of known acid-base transporters on pHi. Under these conditions, halting dialysis unmasked a slow phi decrease caused at least in part by acid-base transport we refer to as "base effiux." Replacing K § in the DF with either NMDG § or TEA + significandy reduced base effiux and made membrane voltage (Vm) more positive. Base effiux in K+-dialyzed axons was stimulated by decreasing the pH of the ASW (pHo) from 8 to 7, implicating transport of acid or base. Although postdialysis acidifications also occurred in axons in which we replaced the K + in the DF with Li +, Na +, Rb +, or Cs +, only with Rb + was base efflux stimulated by low pHo. Thus, the base effiuxes supported by K § and Rb § appear to be unrelated mechanistically to those observed with Li § Na § or Cs § The combination of 437 mM K § and 12 mM HCO~ in the ASW, which eliminates the gradient favoring a hypothetical K+/HCO~ effiux, blocked pHi recovery in K+-dialyzed axons. However, the pHi recovery was not blocked by the combination of 437 mM Na +, veratridine, and CO2/HCO~ in the ASW, a treatment that inverts electrochemical gradients for H § and HCOg and would favor passive H § and HCOg fluxes that would have alkalinized the axon. Similarly, the recovery was not blocked by K § alone or HCOg alone in the ASW, nor was it inhibited by the K-H pump blocker Sch28080 nor by the Na-H exchange inhibitors amiloride and hexamethyleneamiloride. Our data suggest that a major component of base effiux in alkali-loaded axons cannot be explained by metabolism, a H + or HCO~ conductance, or by a K-H exchanger. However, this component could be mediated by a novel K/HCO~ cotransporter.

Cloning and characterization of an electrogenic Na/HCO3− cotransporter from the squid giant fiber lobe

“…This finding is intriguing because, in the squid giant axon, replacing Na ϩ with Li ϩ abolishes NDCBE activity (7). We test K ϩ , because previous investigators described K/HCO 3 Ϫ cotransport activity in the squid giant axon (30,31), and the molecular identity of the transporter responsible for that activity is still not known.…”

“…For example, in the squid giant axon, researchers demonstrated that recovery of intracellular pH (pH i ) from an acid load is mediated by an electroneutral transporter that is: 1) blocked by stilbene derivates such as SITS (7), and 2) transports Na ϩ and the equivalent of 2 HCO 3 Ϫ into the cell in exchange for an intracellular Cl Ϫ (7). Physiological evidence from squid axons also suggests that pH i recovery from an alkali load is mediated by another electroneutral transporter that is inhibited by quaternary ammonium ions (20)-not stilbene derivatives-and mediates the cotransport of K ϩ and HCO 3 Ϫ (30,51).…”

Sodium-Coupled Bicarbonate Transporters

“…However, we found that the pH i recovery (i.e., a decrease in pH i ) from alkaline loads by rat neurons or astrocytes is neither Cl -dependent nor DIDS sensitive. The molecular substrate of this pH i recovery may be the K/HCO 3 cotransporter, in squid axons [35,36,37]. K/HCO 3 cotransporter is not blocked by DIDS, but by quaternary amines.…”

Transport activities involved in intracellular pH recovery following acid and alkali challenges in rat brain microvascular endothelial cells

“…The lack of effect of high K + solution on the initial rate of recovery rules out K + -dependent acid influx through a putative K + /H + exchanger [21] or a K þ HCO À 3 cotransporter [16]. The lack of effect of high K + and of the K + channel blockers, Ba 2+ and Cs + , further suggests that the acid-loading mechanisms are voltage independent as each of these replacements or inhibitors is expected to produce a marked depolarization of the cells.…”