Back
Acta Physiologica 2012; Volume 204, Supplement 689
91st Annual Meeting of The German Physiological Society
3/22/2012-3/25/2012
Dresden, Germany
THE SUBUNIT CONFIGURATION ( VERSUS ) OF THE HUMAN EPITHELIAL SODIUM CHANNEL AFFECTS ITS REGULATION BY THE SERUM- AND GLUCOCORTICOID-INDUCIBLE KINASE (TYPE 1)
Abstract number: O48
Diakov1 *A., Haerteis1 S., Korbmacher1 C.
1Friedrich-Alexander-Universitt Erlangen-Nrnberg, Institut fr Zellulre und Molekulare Physiologie, Erlangen, Germany
Question:
The human epithelial sodium channel (ENaC) may assemble as an abg- or a dbg-trimer. Subunit composition is known to affect ENaC function and may also modify channel regulation e.g. via kinases. In the present study we investigated the effect of SGK1 on human abg-ENaC and dbg-ENaC.
Methods:
Human abg-ENaC or dbg-ENaC was heterologously expressed in Xenopus laevis oocytes. ENaC activity was monitored by measuring the amiloride-sensitive current (DIami) in whole oocytes using the two-electrode voltage-clamp technique and in excised outside-out patches using the patch-clamp technique. A chemiluminescence assay with a FLAG-tagged b-subunit was used to determine surface expression of ENaC.
Results:
Co-expression of constitutively active SGK1 increased whole-cell DIami and surface expression in both abg- and dbg-ENaC expressing oocytes. In contrast, recombinant active SGK1 increased DIami in outside-out patches from oocytes expressing abg-ENaC but not in those from oocytes expressing dbg-ENaC. As expected, an alanine to serine exchange (S594A) in a conserved SGK-consensus motif in the C-terminus of a-ENaC abolished the stimulatory effect of SGK1 on abg-ENaC. Interestingly, this motif is absent in d-ENaC. A chimeric d/a-subunit (da-chim) was generated in which part of the second transmembrane domain and the entire C-terminus of the d-subunit was substituted with the corresponding parts of the a-subunit. We demonstrated that da-chimbg-ENaC was functional with single-channel properties similar to those of dbg-ENaC. However, in outside-out patches SGK1 failed to activate da-chimbg-ENaC.
Conclusions:
Co-expression of SGK1 stimulates both human abg-ENaC and dbg-ENaC by increasing channel surface expression. In contrast, in outside-out patches SGK1 acutely stimulates abg-ENaC but not dbg-ENaC which lacks a conserved SGK-consensus motif in its C-terminus. Interestingly, replacing the C-terminus of d-ENaC with that of a-ENaC is not sufficient to establish an acute stimulatory effect of SGK1 in outside-out patches.
To cite this abstract, please use the following information:
Acta Physiologica 2012; Volume 204, Supplement 689 :O48