Protein kinase regulation of a cloned epithelial Na+ channel

Academic Article


  • 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 <20% of that previously observed. In vitro-synthesized αβγ-rENaC formed an amiloride-sensitive Na -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. + +
  • Published In

    Digital Object Identifier (doi)

    Author List

  • Awayda MS; Ismailov II; Berdiev BK; Fuller CM; Benos DJ
  • Start Page

  • 49
  • End Page

  • 65
  • Volume

  • 108
  • Issue

  • 1