Sodium͞hydrogen exchangers (NHEs) are ubiquitous ion transporters that serve multiple cell functions. We have studied two mammalian isoforms, NHE1 (ubiquitous) and NHE3 (epithelial-specific), by measuring extracellular proton (H ؉ ) gradients during whole-cell patch clamp with perfusion of the cell interior. Maximal Na ؉ -dependent H ؉ fluxes (JH؉) are equivalent to currents >20 pA for NHE1 in Chinese hamster ovary fibroblasts, >200 pA for NHE1 in guinea pig ventricular myocytes, and 5-10 pA for NHE3 in opossum kidney cells.
Sodium͞hydrogen exchangers (NHEs) are present in simple prokaryotes, lower eukaryotes, and higher eukaryotes including plants, fungi, and animals (1). In prokaryotes, yeast, and plants, the driving force of ⌬H ϩ energizes NHEs to export Na ϩ from the cytosol, whereas in animal cells, ⌬Na ϩ drives NHEs to extrude H ϩ . In mammals, eight NHE isoforms (NHE1-NHE8) are cloned (1). NHE1 is a ubiquitous plasma membrane transporter that regulates pH and cell volume. It is implicated in regulating diverse cellular functions including proliferation, adhesion, and migration (2-4). NHE3 is present on endosomal and plasma membranes of epithelial cells, in which it plays a pivotal role in transepithelial Na ϩ transport in multiple organs (5, 6). The functions of other isoforms, present on both cell-surface (NHE2, NHE4, and NHE8) and organellar (NHE6 and NHE7) membranes, remain largely undefined (7,8).NHE activities are typically determined by changes in bulk cytoplasmic pH by using fluorescent pH-sensitive dyes, Na 22 isotopic flux, or radioactive͞fluorescent membrane-permeant buffer trapping. A common protocol is to acidify the cytoplasm and then quantify acid extrusion by NHEs under near V max conditions during Na ϩ -induced pH recovery. Pervasive problems of this approach include low sensitivity, poor time resolution, changes of ion concentrations, and very limited control of the cytoplasmic milieu. Here we describe an approach using pH microelectrodes during whole-cell patch clamp recording. Extracellular H ϩ gradients caused by H ϩ transport are detected by moving cells close to and away from a pH microelectrode in the presence of low buffer concentrations and measuring the H ϩ gradient with cancellation of electrode drift (9-11). Extensive manipulation of the cytoplasmic medium is possible by pipette perfusion, and H ϩ fluxes can be quantified by simulation of buffer diffusion. Using this ''self-referencing'' pH microelectrode technique, we provide different perspectives on the regulatory properties of NHE1 and NHE3 isoforms, in particular their lipid-and mechanosensitive properties. Specifically, we demonstrate that individual phosphatidylinositides have rapid, specific effects on NHE1 and NHE3 and that NHE1 can be very sensitive to rearrangements of its surrounding lipid domain. However, hydrophobic mismatch does not seem to be the primary molecular signal that mediates NHE1 volume sensing.
MethodsCell Culture. Chinese hamster ovary (CHO) fibroblasts were maintained in F-12K nutrient mixture, Kai...