Amiloride-sensitive sodium absorption is different in vertebrates and invertebrates

Sobczak Katja, Willing Anne, Kusche Kristina, Bangel Nadine, Weber Wolf-Michael

Abstract

Amiloride-sensitive Na+ absorption is a well-described feature ofnumerous transporting epithelia in vertebrates. Yet, very little isknown 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 thedivalent 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).