Protein kinase regulation of a cloned epithelial Na+ channel

Mouhamed S. Awayda, Iskander I. Ismailov, Bakhram K. Berdiev, Catherine M. Fuller, Dale J. Benos

Research output: Contribution to journalArticle

78 Citations (Scopus)

Abstract

We examined the regulation of a cloned epithelial Na+ channel (αβγ- rENaC) by protein kinase A (PKA) and protein kinase C (PKC). Experiments were performed in Xenopus oocytes and in planar lipid bilayers. At a holding potential of -100 mV, amiloride-sensitive current averaged -1,279 ± 111 nA (n = 7) in αβγ-rENaC-expressing oocytes. Currents in water-injected oocytes were essentially unresponsive to 10 μM amiloride. A 1-h stimulation of PKC with 100 nM of PMA inhibited whole-cell currents in Xenopus oocytes to 17.1 ± 1.8, and 22.1 ± 2.6% of control (n = 7), at holding potentials of - 100 and +40 mV, respectively. Direct injection of purified PKC resulted in similar inhibition to that observed with PMA. Additionally, the inactive phorbol ester, phorbol-12-myristate-13-acetate, 4-O-methyl, was without effect on αβγ-rENaC currents. Pretreatment with the microtubule inhibitor colchicine (100 μM) did not modify the inhibitory effect of PMA; however, pretreatment with 20 μM cytochalasin B decreased the inhibitory action of PMA to +-selective channel when incorporated into planar lipid bilayers. Addition of PKC, diacyl-glycerol, and Mg-ATP to the side opposite that which amiloride blocked, decreased the channel's open probability (P(o)) from 0.44 ± 0.06 to 0.13 ± 0.03 (n = 9). To study the effects of PKA on αβγ-rENaC expressed in Xenopus oocytes, cAMP levels were elevated with 10 μM forskolin and 1 mM isobutyl-methyl-xanthine. This cAMP- elevating cocktail did not cause any stimulation of αβγ-rENaC currents in either the inward or outward directions. This lack of activation was also observed in oocytes preinhibited with PMA and in oocytes pretreated with cytochalasin B and PMA. Neither α-rENaC nor αβγδ-rENaC incorporated into planar lipid bilayers could be activated with PKA and Mg-ATP added to either side of the membrane, as P(o) remained at 0.63 ± 0.06 (n = 7) and 0.45 ± 0.05 (n = 9), respectively. We conclude that: αβγ-rENaC is inhibited by PKC, and that αβγ-rENaC is not activated by PKA.

Original languageEnglish
Pages (from-to)49-65
Number of pages17
JournalJournal of General Physiology
Volume108
Issue number1
DOIs
Publication statusPublished - Jul 1996
Externally publishedYes

Fingerprint

Epithelial Sodium Channels
Protein Kinases
Oocytes
Protein Kinase C
Cyclic AMP-Dependent Protein Kinases
Amiloride
Lipid Bilayers
Xenopus
Cytochalasin B
Adenosine Triphosphate
Xanthine
Colchicine
Phorbol Esters
Colforsin
Microtubules
Glycerol
Acetates
Injections
Membranes
Water

Keywords

  • ENaC
  • oocytes
  • PKA
  • PKC
  • PLB

ASJC Scopus subject areas

  • Physiology

Cite this

Awayda, M. S., Ismailov, I. I., Berdiev, B. K., Fuller, C. M., & Benos, D. J. (1996). Protein kinase regulation of a cloned epithelial Na+ channel. Journal of General Physiology, 108(1), 49-65. https://doi.org/10.1085/jgp.108.1.49

Protein kinase regulation of a cloned epithelial Na+ channel. / Awayda, Mouhamed S.; Ismailov, Iskander I.; Berdiev, Bakhram K.; Fuller, Catherine M.; Benos, Dale J.

In: Journal of General Physiology, Vol. 108, No. 1, 07.1996, p. 49-65.

Research output: Contribution to journalArticle

Awayda, MS, Ismailov, II, Berdiev, BK, Fuller, CM & Benos, DJ 1996, 'Protein kinase regulation of a cloned epithelial Na+ channel', Journal of General Physiology, vol. 108, no. 1, pp. 49-65. https://doi.org/10.1085/jgp.108.1.49
Awayda MS, Ismailov II, Berdiev BK, Fuller CM, Benos DJ. Protein kinase regulation of a cloned epithelial Na+ channel. Journal of General Physiology. 1996 Jul;108(1):49-65. https://doi.org/10.1085/jgp.108.1.49
Awayda, Mouhamed S. ; Ismailov, Iskander I. ; Berdiev, Bakhram K. ; Fuller, Catherine M. ; Benos, Dale J. / Protein kinase regulation of a cloned epithelial Na+ channel. In: Journal of General Physiology. 1996 ; Vol. 108, No. 1. pp. 49-65.
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