The cystic fibrosis transmembrane conductance regulator (CFTR) consists of five domains, two transmembrane-spanning domains, each composed of six transmembrane segments, a regulatory domain, and two nucleotide-binding domains (NBDs). CFTR is expressed in kidney, but its role in overall renal function is not well understood, because mutations in CFTR found in patients with cystic fibrosis are not associated with renal dysfunction. To learn more about the distribution and functional forms of CFTR in kidney, we used a combination of molecular, cell biological, and electrophysiological approaches. These include an evaluation of CFTR mRNA and protein expression, as well as both two-electrode and patch clamping of CFTR expressed either in Xenopus oocytes or mammalian cells. In addition to wild-type CFTR mRNA, an alternate form containing only the first transmembrane domain (TMD), the first NBD, and the regulatory domain (TNR-CFTR) is expressed in kidney. Although missing the second set of TMDs and the second NBD, when expressed in Xenopus oocytes, TNR-CFTR has cAMP-dependent protein kinase A (PKA)-stimulated single Cl- channel characteristics and regulation of PKA activation of outwardly rectifying Cl- channels that are very similar to those of wild-type CFTR. TNR-CFTR mRNA is produced by an unusual mRNA processing mechanism and is expressed in a tissue-specific manner primarily in renal medulla.
Recordings of single potassium channels from the apical membrane of rabbit cortical collecting tubule have been achieved using the patch-clamp technique. The conductive properties of the channel have been studied in insideout patches. The slope conductance of the open channel is -90 pS. The channel is selective to potassium over sodium, with a selectivity ratio of 9:1. Decreasing the calcium concentration of the solution bathing the cytoplasmic face of the patch results in a decrease of the open-channel probability. Decreasing the calcium concentration to 10 nM or less completely inhibited channel activity. The channel is also inhibited by barium in a dose-dependent fashion.The initial and cortical collecting tubules are the major sites of potassium secretion by the kidney, and it appears that a passive transport mechanism, located in the apical membrane, can account for this translocation process (1). Evidence from perfused tubule studies suggests that there is a barium-sensitive potassium conductance in the apical membrane of the rabbit cortical collecting tubule (2-4).The objective of the present study was to examine this potassium conductance in the apical membrane by singlechannel analysis.Using the well established techniques of manual dissection and isolation of individual tubules from rabbit kidneys (5), we were able to expose the apical membranes of the cells by tearing the tubule wall (6). We have been able to demonstrate the existence in these cells of barium-sensitive potassium channels that are calcium-activated.
MATERIALS AND METHODSCortical collecting tubule segments (-1 mm long) were isolated from the kidneys of female New Zealand White rabbits by manual dissection (5). The apical membrane was exposed by ripping the tubule lengthwise with a sharp needle. The tubule was then secured by means of two strips of aluminum foil, which were wrapped around a small glass coverslip (=12 x 4 mm). The tubule, together with the coverslip, was transferred to a chamber, containing a Hepes-buffered ringer (pH, 7.3), mounted on a microscope stage. The composition of this bath solution was 135 mM sodium/5 mM potassium/1.8 mM calcium/1 mM magnesium/145.6 mM chloride/10 mM Hepes/2 mM glucose. The tubule was then examined at a magnification of x400 using Hoffman optics, and the apical surface of the cells was identified. The apical membrane of a cell was approached with a patch pipette under micromanipulator control and high-resistance seals (>10 G Ql) were generally achieved spontaneously (without suction).The patch pipettes always contained a filtered solution of the following composition: 75 mM sodium/75 mM potassium/1.8 mM calcium/1 mM magnesium/10 mM Hepes/155.6 mM chloride (adjusted to pH 7.3 with NaOH). When potassium channels were present in the patch, they were seen to open and close spontaneously. The pipette was then withdrawn from the cell, bringing a small patch of membrane with it. If the patch showed signs of vesicle formation [loss of channel activity or distortion of channel currents (7)], it ...
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