The principal cell of the kidney collecting duct is one of the most highly regulated epithelial cell types in vertebrates. The effects of hormonal, autocrine, and paracrine factors to regulate principal cell transport processes are central to the maintenance of fluid and electrolyte balance in the face of wide variations in food and water intake. In marked contrast with the epithelial cells lining the proximal tubule, the collecting duct is electrically tight, and ion and osmotic gradients can be very high. The central role of principal cells in salt and water transport is reflected by their defining transporters-the epithelial Na 1 channel (ENaC), the renal outer medullary K 1 channel, and the aquaporin 2 (AQP2) water channel. The coordinated regulation of ENaC by aldosterone, and AQP2 by arginine vasopressin (AVP) in principal cells is essential for the control of plasma Na 1 and K 1 concentrations, extracellular fluid volume, and BP. In addition to these essential hormones, additional neuronal, physical, and chemical factors influence Na 1 , K 1 , and water homeostasis. Notably, a variety of secreted paracrine and autocrine agents such as bradykinin, ATP, endothelin, nitric oxide, and prostaglandin E 2 counterbalance and limit the natriferic effects of aldosterone and the water-retaining effects of AVP. Considerable recent progress has improved our understanding of the transporters, receptors, second messengers, and signaling events that mediate principal cell responses to changing environments in health and disease. This review primarily addresses the structure and function of the key transporters and the complex interplay of regulatory factors that modulate principal cell ion and water transport.

Reduction of NDPs by murine ribonucleotide reductase (mRR) requires catalytic (mR1) and free radical-containing (mR2) subunits and is regulated by nucleoside triphosphate allosteric effectors. Here we present a new, comprehensive, and quantitative model for allosteric control of mRR enzymatic activity based on molecular mass, ligand binding, and enzyme activity studies. In this model, nucleotide binding to the specificity site (s-site) drives formation of an active R1(2)R2(2) dimer, ATP or dATP binding to the adenine-specific site (a-site) results in formation of an inactive tetramer, and ATP binding to the newly described hexamerization site (h-site) drives formation of active R1(6)R2(6) hexamer. In contrast, an earlier phenomenological model [Thelander, L., and Reichard, P. (1979) Annu. Rev. Biochem. 67, 71-98] (the "RT" model) ignores aggregation state changes and mistakenly rationalizes ATP activation versus dATP inhibition as reflecting different functional consequences of ATP versus dATP binding to the a-site. Our results suggest that the R1(6)R2(6) heterohexamer is the major active form of the enzyme in mammalian cells, and that the ATP concentration is the primary modulator of enzyme activity, coupling the rate of DNA biosynthesis with the energetic state of the cell. Using the crystal structure of the Escherichia coliR1 hexamer as a model for the mR1 hexamer, a scheme is presented that rationalizes the slow isomerization of the tetramer form and suggests an explanation for the low enzymatic activity of tetramers complexed with R2. The similar specific activities of R1(2)R2(2) and R1(6)R2(6) are inconsistent with a proposed model for R2(2) docking with R1(2) [Uhlin, U., and Eklund, H. (1994) Nature 370, 533-539], and an alternative is suggested.

Our understanding of epithelial Na(+) channel (ENaC) structure and function has been profoundly impacted by the resolved structure of the homologous acid-sensing ion channel 1 (ASIC1). The structure of the extracellular and pore regions provide insight into channel assembly, processing, and the ability of these channels to sense the external environment. The absence of intracellular structures precludes insight into important interactions with intracellular factors that regulate trafficking and function. The primary sequences of ASIC1 and ENaC subunits are well conserved within the regions that are within or in close proximity to the plasma membrane, but poorly conserved in peripheral domains that may functionally differentiate family members. This review examines functional data, including ion selectivity, gating, and amiloride block, in light of the resolved ASIC1 structure.

ELIC, the pentameric ligand-gated ion channel from Erwinia chrysanthemi, is a prototype for Cys-loop receptors. Here we show that acetylcholine is a competitive antagonist for ELIC. We determine the acetylcholine–ELIC cocrystal structure to a 2.9-Å resolution and find that acetylcholine binding to an aromatic cage at the subunit interface induces a significant contraction of loop C and other structural rearrangements in the extracellular domain. The side chain of the pore-lining residue F247 reorients and the pore size consequently enlarges, but the channel remains closed. We attribute the inability of acetylcholine to activate ELIC primarily to weak cation-π and electrostatic interactions in the pocket, because an acetylcholine derivative with a simple quaternary-to-tertiary ammonium substitution activates the channel. This study presents a compelling case for understanding the structural underpinning of the functional relationship between agonism and competitive antagonism in the Cys-loop receptors, providing a new framework for developing novel therapeutic drugs.

Reduction of NDPs by murine ribonucleotide reductase (mRR) requires catalytic (mR1) and free radical-containing (mR2) subunits and is regulated by nucleoside triphosphate allosteric effectors. Here we present the results of several studies that refine the recently presented comprehensive model for the allosteric control of mRR enzymatic activity [Kashlan, O. B., et al. (2002) Biochemistry 41, 462-474], in which nucleotide binding to the specificity site (s-site) drives formation of an active R1(2)R2(2) dimer, ATP or dATP binding to the adenine site (a-site) drives formation of a tetramer, mR1(4a), which isomerizes to an inactive form, mR1(4b), and ATP binding to the hexamerization site (h-site) drives formation of an active R1(6)R2(6) hexamer. Analysis of the a-site D57N variant of mR1, which differs from wild-type mR1 (wt-mR1) in that its RR activity is activated by both ATP and dATP, demonstrates that dATP activation of the D57N variant RR arises from a blockage in the formation of mR1(4b) from mR1(4a), and provides strong evidence that mR1(4a) forms active complexes with mR2(2). We further demonstrate that (a) differences in the effects of ATP versus dATP binding to the a-site of wt-mR1 provide specific mechanisms by which the dATP/ATP ratio in mammalian cells could modulate in vivo RR enzymatic activity, (b) the comprehensive model is valid over a range of Mg(2+) concentrations that include in vivo concentrations, and (c) equilibrium constants derived for the comprehensive model can be used to simulate the distribution of R1 among dimer, tetramer, and hexamer forms in vivo. Such simulations indicate that mR1(6) predominates over mR1(2) in the cytoplasm of normal mammalian cells, where the great majority of RR activity is located, but that mR1(2) may be important for nuclear RR activity and for RR activity in cells in which the level of ATP is depleted.