Permeability properties of ENaC selectivity filter mutants.

The epithelial Na(+) channel (ENaC), located in the apical membrane of tight epithelia, allows vectorial Na(+) absorption. The amiloride-sensitive ENaC is highly selective for Na(+) and Li(+) ions. There is growing evidence that the short stretch of amino acid residues (preM2) preceding the putative second transmembrane domain M2 forms the outer channel pore with the amiloride binding site and the narrow ion-selective region of the pore. We have shown previously that mutations of the alphaS589 residue in the preM2 segment change the ion selectivity, making the channel permeant to K(+) ions. To understand the molecular basis of this important change in ionic selectivity, we have substituted alphaS589 with amino acids of different sizes and physicochemical properties. Here, we show that the molecular cutoff of the channel pore for inorganic and organic cations increases with the size of the amino acid residue at position alpha589, indicating that alphaS589 mutations enlarge the pore at the selectivity filter. Mutants with an increased permeability to large cations show a decrease in the ENaC unitary conductance of small cations such as Na(+) and Li(+). These findings demonstrate the critical role of the pore size at the alphaS589 residue for the selectivity properties of ENaC. Our data are consistent with the main chain carbonyl oxygens of the alphaS589 residues lining the channel pore at the selectivity filter with their side chain pointing away from the pore lumen. We propose that the alphaS589 side chain is oriented toward the subunit-subunit interface and that substitution of alphaS589 by larger residues increases the pore diameter by adding extra volume at the subunit-subunit interface.

Biogeography of Sulawesian shrews: testing for their origin with a parametric bootstrap on molecular data.

In order to identify the zoogeographic origin of shrews (genus Crocidura) living on the oceanic island of Sulawesi, 15 taxa from Southeast Asia and 1 from Europe were examined for sequence variation in a segment (617 bp) of the mitochondrial cytochrome b gene. The null hypothesis of a monophyletic origin of all Sulawesian shrews was investigated by a phylogenetic reconstruction using maximum parsimony. According to a parametric bootstrap which simulated sequence evolution for these taxa, the null hypothesis could be rejected as highly unlikely (P < 0.01). Therefore, the molecular phylogeny strongly suggests that overwater colonization of Sulawesi by shrews succeeded on at least two occasions. The first, relatively ancient wave of colonizers radiated and gave rise to a surprizingly diverse assemblage of at least five species which now coexist in perfect sympatry on Sulawesi. The second wave, of more recent origin, gave rise to Crocidura nigripes, a species which retained close genetic affinities with other Malay shrews.