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We examined the properties of Na + /K + /2Cl − cotransport in cultured mouse mTAL cells with respect to its kinetics, the contribution of K/K exchange to K fluxes mediated by the cotransporter, and [ 3 H]bumetanide binding and turnover numbers in media with varying osmolality. The addition of bumetanide, the replacement of external Na + or the replacement of external Cl − resulted in an almost identical (approx. 50%) decrease in K + influx, suggesting that Na + -dependent, Cl − -dependent, BS K + influx was a measure of Na + /K + /2Cl − cotransport. The kinetics of the BS K + influx revealed a high affinity for external Na + ( apparent K m 7 mM ) and external K + ( apparent K m 1.3 mM ), but a very low affinity for external Cl − apparent K m 67 mM with a two-site model ). Of interest was the finding that none of the K + ( 86 RB + ) efflux was sensitive to bumetanide, suggesting the absence of cotransport mediated K/K exchange in this cell type. Specific [ 3 H]bumetanide binding was a saturable function of free bumetanide concentration with a K d of 0.20 μ M and maximum binding ( B max ) of 0.63 pmol/mg, or about 53 000 sites per cell. Simultaneous transport and bumetanide binding assays yielded a turnover number of 255 min −1 . The omission of external Na + , K + or Cl − reduced specific [ 3 H]bumetanide binding to values indistinguishable from zero. Changing medium osmolarity resulted in a co-ordinate change in BS K + influx and bumetanide binding, with a monotonic increase in both transport and bumetanide binding with increase in osmolality from 200 to 400 mosmol/kg. About 85% of the cotransporter sites were located on the apical side, as in the intact mTAL tubule. The simultaneous measurement of BS ion transport and [ 3 H]bumetanide binding in the mTAL cell may provide valuable insights into the regulation of Na + / K + /2Cl − cotransport in this nephron segment.

The K-Cl cotransporter protein KCC1 is a membrane transport protein that mediates the coupled, electroneutral transport of K and Cl across plasma membranes. The precise cell type(s) in the kidney that express the K-Cl cotransporter have remained unknown. The aim of the present investigation was to define the distribution of KCC1 mRNA in the human kidney. We used in situ hybridization with a nonradioactive digoxigenin-labeled riboprobe. We identified abundant KCC1 mRNA expression in the epithelial cells throughout the distal and proximal renal tubular epithelium. The transporter was also expressed in glomerular mesangial cells and endothelial cells of the renal vessels. These findings suggest that the K-Cl cotransporter may have an important role in transepithelial K and Cl reabsorption.

Among the many intellectual challenges encountered by medical students, residents, and practitioners, that of interpreting clinical acid-base disorders remains one of the most difficult challenges to master. This short book is an attempt "to lead the uninitiated and the timid to an understanding of acid-base disorders." The author was encouraged to write yet another book on the subject of acid-base disorders because of "explosion in knowledge regarding the molecular basis for membrane transport" in the last decade. To acquaint the beginning reader with the basic molecular mechanisms, the author provides an excellent, albeit brief, description of the importance of pH in life and the role played by various primary and secondary active transport pathways involved in regulation of intracellular pH. The discussion of transport pathways involved in proton secretion is laudable and unusual for a short primer and accounts for the strength of the book. I gave a copy to

The majority of the ouabain-insensitive K influx in human erythrocytes is dependent on the presence of Cl. Recent studies have shown that a portion of the Cl-dependent K influx persists in the absence of external Na (Nao). It has been suggested that this Nao-independent component represents (K + Cl) cotransport, whereas the remainder of the Cl-dependent K influx seen on addition of external Na represents (Na + K + 2Cl) cotransport. In the present studies, the kinetics of Cl-dependent K influx were examined in the presence and absence of external Na, by varying external K and external Cl. Our studies suggest that the Nao-independent Cl-dependent pathway has a relatively low affinity for external K (Km 17-30 mM) in contrast to the high affinity of the Nao-augmented component (Km 3-4 mM). N-ethylmaleimide (NEM) stimulates the maximal velocity of the Nao-independent Cl-dependent K influx achievable without alteration of intracellular solutes but does not alter its Km for external K. In contrast, NEM has no stimulatory effect on the Nao-augmented component. The Cl dependence of the Nao-independent K influx is best described by a relatively flat curve with a mild upward concavity. The kinetic properties of the Nao-independent component of Cl-dependent K transport are very similar to those of the putative (K + Cl) cotransport pathway seen in low-K sheep erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)