Inverse Relationship Between Membrane Lipid Fluidity and Activity of Na + /H + Exchangers, NHE1 and NHE3, in Transfected Fibroblasts

Abstract

This report presents a study of the effects of the membrane fluidizer, benzyl alcohol, on NHE isoforms 1 and 3. Using transfectants of an NHE-deficient fibroblast, we analyzed each isoform separately. An increase in membrane fluidity resulted in a decrease of approximately 50% in the specific activities of both NHE1 and NHE3. Only Vmax was affected; KNa was unchanged. This effect was specific, as Na+, K+, ATPase activity was slightly stimulated. Inhibition of NHE1 and NHE3 was reversible and de novo protein synthesis was not required to restore NHE activity after washout of fluidizer. Inhibition kinetics of NHE1 by amiloride, 5-(N,N-dimethyl)amiloride (DMA), 5-(N-hexamethyl)amiloride (HMA) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were largely unchanged. Half-maximal inhibition of NHE3 was also reached at approximately the same concentrations of amiloride and analogues in control and benzyl alcohol treated, suggesting that the amiloride binding site was unaffected. Inhibition of vesicular transport by incubation at 4 degrees C augmented the benzyl alcohol inhibition of NHE activity, suggesting that the fluidizer effect does not solely involve vesicle trafficking. In summary, our data demonstrate that the physical state of membrane lipids (fluidity) influences Na+/H+ exchange and may represent a physiological regulatory mechanism of NHE1 and NHE3 activity.

Regulation of Na+/H+ exchanger 1 allosteric balance by its localization in cholesterol‐ and caveolin‐rich membrane microdomains

“…One might then propose that NHE-1 allosteric transition to the high affinity form may be enhanced by an increase in the fluidity of its membrane microenvironnement (i.e., in lipid microdomains with a reduced content in cholesterol or outside cholesterol-rich microdomains). However, an increase in membrane fluidity has previously been shown to decrease NHE-1 V max of transport for Na þ without any change in K Na (Bookstein et al, 1997). By contrast we found no detectable change in V max in our experiments.…”

“…However, few studies have considered the influence of membrane lipids on its activity. Membrane fluidity has been identified as one such modulator (Bookstein et al, 1997;Gorria et al, 2006). Recently, we have obtained evidence that osmotic regulation modifies the allosteric balance of NHE-1 and that membrane curvature and/or tension would by itself modulate NHE-1 (Lacroix et al, unpublished work).…”

“…Besides its role in proliferation processes, NHE-1 activation has also been reported to be part of apoptotic processes upon diverse stimuli (Khaled et al, 2001;Huc et al, 2004Huc et al, , 2007Lagadic-Gossmann et al, 2004. In this context, how an NHE-1 activation of similar amplitude plays a role in such opposite processes remains to be elucidated and might stem from different regulation pathways, possibly resulting from differential phosphorylation (Khaled et al, 2001;Bourguignon et al, 2004;Mukhin et al, 2004), binding of modulators (Denker et al, 2000;Garnovskaya et al, 2003), or merely membrane characteristic changes (Bookstein et al, 1997).…”

Effects of temperature on intracellular sodium, pH and cellular energy status in RIF‐1 tumor cells

“…A change in membrane fluidity could also affect the kinetics of membrane embedded proteins such as Na þ /K þ -ATPase and other ion transporters. 22,23 Temperature could also alter the activity of ion transport proteins directly and hence change Na þ i concentration. 24,25 Another possibility is that a change in temperature alters Na þ i to maintain the homeostasis of other ions.…”

“…In this study we have investigated the effects of increasing the temperature from 33 to 37 C on Na þ i , cellular energy status, pH i and pH e in perfused RIF-1 cells. We employed 23 Na NMR spectroscopy and the paramagnetic shift reagent (SR), thulium (III) 1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetrakis (methylene phosphonate) (TmDOTP À5 ), to monitor Na þ i , and 31 P NMR spectroscopy to monitor cellular energy status and pH. We also investigated the effects of 5-(N-ethyl-Nisopropyl) amiloride (EIPA), a potent and specific inhibitor of Na þ /H þ antiporter, to determine the mechanisms of the changes in Na þ i and pH i with temperature.…”