The role of transmembrane domain 2 in cation transport by the Na–K–Cl cotransporter

The human and shark Na–K–Cl cotransporters (NKCC), although 74% identical in amino acid sequence, exhibit marked differences in ion transport and bumetanide binding. We have utilized shark–human chimeras of NKCC1 to search for regions that confer the kinetic differences. Two chimeras (hs3.1 and its reverse sh3.1) with a junction point located at the beginning of the third transmembrane domain were examined after stable transfection in HEK-293 cells. Each carried out bumetanide-sensitive 86Rb influx with cation affinities intermediate between shark and human cotransporters. In conjunction with the previous finding that the N and C termini are not responsible for differences in ion transport, the current observations identify the second transmembrane domain as playing an important role. Site-specific mutagenesis of two pairs of residues in this domain revealed that one pair is indeed involved in the difference in Na affinity, and a second pair is involved in the difference in Rb affinity. Substitution of the same residues with corresponding residues from NKCC2 or the Na-Cl cotransporter resulted in cation affinity changes, consistent with the hypothesis that alternative splicing of transmembrane domain 2 endows different versions of NKCC2 with unique kinetic behaviors. None of the changes in transmembrane domain 2 was found to substantially affect Km(Cl), demonstrating that the affinity difference for Cl is specified by the region beyond predicted transmembrane domain 3. Finally, unlike Cl, bumetanide binding was strongly affected by shark–human replacement of transmembrane domain 2, indicating that the bumetanide-binding site is not the same as the Cl-binding site.

Tree rings, carbon dioxide, and climatic change

Tree rings have been used in various applications to reconstruct past climates as well as to assess the effects of recent climatic and environmental change on tree growth. In this paper we briefly review two ways that tree rings provide information about climate change and CO2: (i) in determining whether recent warming during the period of instrumental observations is unusual relative to prior centuries to millennia, and thus might be related to increasing greenhouse gases; and (ii) in evaluating whether enhanced radial growth has taken place in recent decades that appears to be unexplained by climate and might instead be due to increasing atmospheric CO2 or other nutrient fertilization. It is found that a number of tree-ring studies from temperature-sensitive settings indicate unusual recent warming, although there are also exceptions at certain sites. The present tree-ring evidence for a possible CO2 fertilization effect under natural environmental conditions appears to be very limited.