INVERSE RELATIONSHIP BETWEEN MEMBRANE LIPID FLUIDITY AND ACTIVITY OF NA+ H+ EXCHANGERS, NHE1 AND NHE3, IN TRANSFECTED FIBROBLASTS/

This report presents a study of the effects of the membrane fluidizer, benzyl alcohol, on NHE isoforms 1 and 3. Using transfectants of an NH E-deficient fibroblast, we analyzed each isoform separately. An increa se in membrane fluidity resulted in a decrease of approximate to 50% i n the specific activities of both NHE1 and NHE3. Only V-max was affect ed; K-Na was unchanged. This effect was specific, as Na+, K+, ATPase a ctivity was slightly stimulated. Inhibition of NHE1 and NHE3 was rever sible and de novo protein synthesis was not required to restore NHE ac tivity after washout of fluidizer. Inhibition kinetics of NHE1 by amil oride, 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 a lcohol treated, suggesting that the amiloride binding site was unaffec ted. Inhibition of vesicular transport by incubation at 4 degrees C au gmented 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 lipi ds (fluidity) influences Na+/H+ exchange and may represent a physiolog ical regulatory mechanism of NHE1 and NHE3 activity.