ENaC–Membrane Interactions: Regulation of Channel Activity by Membrane Order

Recently, it was reported that the epithelial Na channel (ENaC) is regulated by temperature (Askwith, C.C., C.J. Benson, M.J. Welsh, and P.M. Snyder. 2001. Proc. Natl. Acad. Sci. USA. 98:6459–6463). As these changes of temperature affect membrane lipid order and lipid–protein interactions, we tested the hypothesis that ENaC activity can be modulated by membrane lipid interactions. Two approaches were used to modulate membrane anisotropy, a lipid order–dependent parameter. The nonpharmacological approach used temperature changes, while the pharmacological one used chlorpromazine (CPZ), an agent known to decrease membrane order, and Gd 3 . Experiments used Xenopus oocytes expressing human ENaC. Methods of impedance analysis were used to determine whether the effects of changing lipid order indirectly altered ENaC conductance via changes of membrane area. These data were further corroborated with quantitative morphology on micrographs from oocytes membranes studied via electron microscopy. We report biphasic effects of cooling (stimulation followed by inhibition) on hENaC conductance. These effects were relatively slow (minutes) and were delayed from the actual bath temperature changes. Peak stimulation occurred at a calculated T max of 15.2. At temperatures below T max , ENaC conductance was inhibited with cooling. The effects of temperature on g Na were distinct from those observed on ion channels endogenous to Xenopus oocytes, where the membrane conductance decreased monoexponentially with temperature (t 6.2 C). Similar effects were also observed in oocytes with reduced intraand extracellular [Na ], thereby ruling out effects of self or feedback inhibition. Addition of CPZ or the mechanosensitive channel blocker, Gd 3 , caused inhibition of ENaC. The effects of Gd 3 were also attributed to its ability to partition into the outer membrane leaflet and to decrease anisotropy. None of the effects of temperature, CPZ, or Gd 3 were accompanied by changes of membrane area, indicating the likely absence of effects on channel trafficking. However, CPZ and Gd 3 altered membrane capacitance in an opposite manner to temperature, consistent with effects on the membrane-dielectric properties. The reversible effects of both Gd 3 and CPZ could also be blocked by cooling and trapping these agents in the rigidified membrane, providing further evidence for their mechanism of action. Our findings demonstrate a novel regulatory mechanism of ENaC. key words: sodium channel • epithelia • Q 10 • impedance analysis • phase transition I N T R O D U C T I O N It has long been known that neuronal ion channels are affected by changes of membrane temperature. These actions of temperature are manifested as complex events of varying magnitude on channel activation, inactivation and unitary conductance (Schwarz, 1979; Rodriguez et al., 1998). In all cases, the channel kinetics and conductance exhibited a positive temperature dependency resulting in acceleration and stimulation at higher temperatures. Recently, it was reported that brain epithelial Na channel homologues (BNaC) exhibited an inverse temperature sensitivity that resulted in stimulation of channel current with cooling to below ambient temperature (Askwith et al., 2001). Moreover, these investigators observed similar effects with hENaC, where temperature changes down to 6 C were found to stimulate currents in Xenopus oocytes clamped to 60 mV. Thus, it appears that ENaC and its homologues exhibit a unique temperature sensitivity not expected from membrane ion channels. These effects of temperature may be of little physiological significance to ENaC function in the majority of mammalian tissues where ENaC resides in a controlled 37 C. However, these findings renew interest in the issues of regulation of ENaC by the membrane environment and tension. Although we have demonstrated that channel activity is not sensitive to direct mechanical perturbations of the membrane (Awayda and Subramanyam, 1998), others have recently found effects of flow on ENaC (Satlin et al., 2001). Moreover, ENaC and its homologues are also found to be components Address correspondence to Mouhamed S. Awayda, Department of Physiology, SL 39, Tulane University Health Sciences Center, New Orleans, LA 70112. Fax: (504)584-2675; email: mawayda@tulane.edu Abbreviations used in this paper: CPZ, chlorpromazine; ENaC, epithelial Na channel. on A uust 6, 2017 jgp.rress. org D ow nladed fom