Amiloride-sensitive sodium absorption is different in vertebrates and invertebrates

Abstract

Amiloride-sensitive Na+absorption is a well-described feature of numerous transporting epithelia in vertebrates. Yet, very little is known about this important physiological process regarding invertebrates. In the present paper, we compare vertebrate Na+absorption mediated by the amiloride-sensitive epithelial Na+channel (ENaC) and its invertebrate counterpart. We used the dorsal skin of the annelid Hirudo medicinalis as a model for the Na+absorption of invertebrate epithelia. In applying electrophysiological, molecular, and biochemical techniques we found striking functional and structural differences between vertebrate and invertebrate amiloride-sensitive Na+absorption. Using modified Ussing chambers, we analyzed the influence of different known blockers and effectors of vertebrate ENaC on leech epithelial Na+absorption. We demonstrate that the serine protease trypsin had no effect on the Na+transport across leech integument, while it strongly activates vertebrate ENaC. While protons, and the divalent cations Ni2+and Zn2+stimulate vertebrate ENaC, amiloride-sensitive Na+currents in leech integument were substantially reduced. For molecular studies, we constructed a cDNA library of Hirudo medicinalis and screened it with specific ENaC antibodies. We performed numerous PCR approaches using a vast number of different degenerated and specific ENaC primers to identify ENaC-like structures. Yet, both strategies did not reveal any ENaC-like sequence in leech integument. From these data we conclude that amiloride-sensitive Na+absorption in leech skin is not mediated by an ENaC-like Na+channel but by a still unknown invertebrate member of the ENaC/DEG family that we termed lENaTP (leech epithelial Na+transporting protein).