Sodium ion-hydrogen ion antiporter: Mechanisms of sodium ion conduction.

Abstract

The Na-H antiporter has been reported to catalyze electroneutral exchange of Na for Na, Na for H, and Na for Li. I have confirmed these findings for the basolateral Na-H antiporter of turtle colon. In addition I have found that in the presence of outwardly directed substrate gradients the Na-H antiporter is also electrogenic, i.e. it mediates net charge flow across the basolateral membrane. Sheets of turtle colon (Pseudemys scripta) were voltage clamped, apically permeabilized with the pore forming polyene antibiotic amphotericin B, and opposing transmural fluxes of $\sp{22}$Na$\sp+$ were determined in the presence of steep Na$\sp+$, Li$\sp+$, or H$\sp+$ gradients. A serosal-to-mucosal (S-to-M) Na$\sp+$ gradient promoted M-to-S $\sp{22}$Na$\sp+$ counterflow at both pH 6.5 and pH 8.5. Serosal amiloride eliminated the trans accelerated Na$\sp+$ flow, consistent with a basolateral localization of the antiporter. No amiloride-sensitive current was detected at either pH, and opposing Na exchange fluxes under these conditions were $\sim$0.2 $\mu$Eq/cm$\sp2\cdot$h. In the presence of an M-to-S Na$\sp+$ gradient S-to-M $\sp{22}$Na$\sp+$ flow was trans accelerated and an outward Na$\sp+$ current was detected. Na-Na counterflow and I$\sb{\rm sc}$ shared a virtually identical sensitivity to amiloride, dimethylamiloride, and ethylisopropylamiloride. Na-Na exchange, Na-H exchange and outward Na current were also identically sensitive to changes in cell volume. These results are consistent with the notion that the Na-H antiporter possesses an electrogenic operating mode in addition to the well known electroneutral operating modes. In the absence of a Na gradient, an outward proton gradient (pH$\sb{\rm i}$ 6.0$\vert$pH$\sb{\rm o}$ 8.5) promoted electrogenic, inward Na$\sp+$ counterflow. Inward proton gradients (pH$\sb{\rm i}$ 8.5$\vert$pH$\sb{\rm o}$ 6.0) promoted outward Na$\sp+$ counterflow but no amiloride sensitive current. Inward Na current driven by outward proton flow is most easily explained by an exchange stoichiometry of 1 proton for $\ge$2 Na. Outward Na current driven by outward Na gradients may also be by this mechanism. Sigmoidal current activation kinetics tend to support this motion. However, we could not exclude a channel-like conformation of the antiporter as an additional (alternative) mechanism mediating outward Na current.