Intracellular Ph Regulation by Na+/H+ Exchange Requires Phosphatidylinositol 4,5-Bisphosphate

Abstract

The carrier-mediated, electroneutral exchange of Na+ for H+ across the plasma membrane does not directly consume metabolic energy. Nevertheless, acute depletion of cellular ATP markedly decreases transport. We analyzed the possible involvement of polyphosphoinositides in the metabolic regulation of NHE1, the ubiquitous isoform of the Na+/H+ exchanger. Depletion of ATP was accompanied by a marked reduction of plasmalemmal phosphatidylinositol 4,5-bisphosphate (PIP2) content. Moreover, sequestration or hydrolysis of plasmalemmal PIP2, in the absence of ATP depletion, was associated with profound inhibition of NHE1 activity. Examination of the primary structure of the COOH-terminal domain of NHE1 revealed two potential PIP2-binding motifs. Fusion proteins encoding these motifs bound PIP2 in vitro. When transfected into antiport-deficient cells, mutant forms of NHE1 lacking the putative PIP2-binding domains had greatly reduced transport capability, implying that association with PIP2 is required for optimal activity. These findings suggest that NHE1 activity is modulated by phosphoinositides and that the inhibitory effect of ATP depletion may be attributable, at least in part, to the accompanying net dephosphorylation of PIP2.

Activation of AMP-activated Protein Kinase Regulates Hippocampal Neuronal pH by Recruiting Na+/H+ Exchanger NHE5 to the Cell Surface

“…In experiments examining the kinetics of NHE5 activity as a function of the extracellular Na ϩ concentration, cells were acid-loaded using the NH 4 ϩ -prepulse technique prior to measuring the influx of 22 Na ϩ as described previously (22). To measure the activity of NHE5 as a function of the intracellular H ϩ (H ϩ i ) concentration, intracellular pH (pH i ) was clamped over the range of 5.4 to 7.4 using the K ϩ -nigericin method (35) as previously described (36). Data analysis and fitting was carried out using Origin 8 software.…”

Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) stimulates the electrogenic Na/HCO 3 cotransporter NBCe1-A expressed in Xenopus oocytes

“…Phosphoinositides also regulate the activity of acid-base transporters. For example, PIP 2 is required for optimal activity of NHE1 transfected into an AP-1 cell line (12), and can rescue reduced NHE1 activity elicited by depleting ATP in Chinese hamster ovary (CHO) fibroblasts (13). Furthermore, the phosphoinositide PIP 3 stimulates NHE3 in opossum kidney cells, but not NHE1 in the CHO cells (13).…”

Mutations of Arg440 and Gly455/Gly456 Oppositely Change pH Sensing of Na+/H+ Exchanger 1

“…Regulation of NHE1 by these stimuli has been reported to occur through a variety of signaling molecules, i.e. calcinuerin B homologous protein (6,7), Ca 2ϩ /calmodulin (8,9), low molecular mass GTPases Ras and Rho (10 -12), p42/44 mitogen-activated protein kinases (13), p90 ribosomal S6 kinase (14), 14-3-3 protein (15), Nck-interacting kinase (16), and phosphatidylinositol 4,5-bisphosphate (17). However, the interrelationship of these signaling molecules and the mechanism by which they modulate the interaction of the putative H ϩ modifier site with intracellular protons is still unclear.…”