Mechanosensitivity of αβγ rENaC expressed in Xenopus oocytes

Academic Article

Abstract

  • A highly sodium selective, amiloride-sensitive Na+ channel (ENaC), structurally similar to the degenerins of C.elegans, a class of proteins associated with mechanosensitivity, has recently been cloned. ENaCs are located in Na+ reabsorbing epithelia that are capable of cell volume regulation, but there is no direct evidence to prove that ENaCs are involved in the regulatory volume decrease (RVD) that occurs following exposure to hypotonicity, or the regulatory volume increase (RVI) after shrinkage. To test the functional response of ENaC to changes in external osmolality, anisosmotic ND-20 media (from 70 mOsM to 450 mOsM) were utilized to superfuse oocytes expressing αβγ rENaC. Whole-cell currents were found to be reversibly dependent on external osmolality. Under conditions of swelling (70 mOsM) or shrinking (450 mOsM), current amplitude decreased to 37.8 ± 6.6 % (n=7) and increased to 162.0 ± 8.6 % (n=8) of control, respectively. In contrast, there was no response of H2O-injected oocytes to the above osmotic stimuli. The osmolality-induced currents were sensitive to amiloride but not to K+ or Cl- channel inhibitors. The Ki of amiloride (Ki =114 ± 0.03 nM, n=5, under isosmotic conditions) was shifted to the right (4.1 ± 1.1 μM, n=4) in swollen cells. In contrast, hyperosmolality enhanced the amiloride sensitivity of αβγ rENaC expressing oocytes (Ki = 55 ± 0.001 nM, n=5). The osmolality-induced Na+ currents showed similar properties to stretch-activated channels, including inhibition by Gd3+ (20 μM) and La3+ (2 mM). The PNa/PK permeation ratios of amiloride-sensitive current in isotonic, hypertonic, and hypotonic media were 20, 11, and 6, respectively (n=8), similar to those of PNa/PCs (33, 12, and 5, respectively, n=6). However, the PNa/PLi ratio was nearly constant (near 1.0, n=5) under all conditions studied. The present study demonstrated that osmotic pressure regulates mechanoactivation of rENaC.
  • Author List

  • Ji HL; Fuller CM; Benos DJ
  • Volume

  • 12
  • Issue

  • 5