Chloride channel and chloride conductance regulator domains of CFTR, the cystic fibrosis transmembrane conductance regulator

CFTR is a cyclic AMP (cAMP)-activated chloride (Cl-) channel and a regulator of outwardly rectifying Cl- channels (ORCCs) in airway epithelia. CFTR regulates ORCCs by facilitating the release of ATP out of cells. Once released from cells, ATP stimulates ORCCs by means of a purinergic receptor. To define the domains of CFTR important for Cl- channel function and/or ORCC regulator function, mutant CFTRs with N- and C-terminal truncations and selected individual amino acid substitutions were created and studied by transfection into a line of human airway epithelial cells from a cystic fibrosis patient (IB3-1) or by injection of in vitro transcribed complementary RNAs (cRNAs) into Xenopus oocytes. Two-electrode voltage clamp recordings, 36Cl- efflux assays, and whole cell patch-clamp recordings were used to assay for the Cl- channel function of CFTR and for its ability to regulate ORCCs. The data showed that the first transmembrane domain (TMD-1) of CFTR, especially predicted alpha-helices 5 and 6, forms an essential part of the Cl- channel pore, whereas the first nucleotide-binding and regulatory domains (NBD1/R domain) are essential for its ability to regulate ORCCs. Finally, the data show that the ability of CFTR to function as a Cl- channel and a conductance regulator are not mutually exclusive; one function could be eliminated while the other was preserved.     

Stimulation of Cl conductance by minoxidil sulfate and K conductance by minoxidil in eccrine clear cells

Minoxidil sulfate (MXS), an antihypertensive agent and hair growth promoter, has been reported to stimulate K channels in vascular smooth muscle cells. We now report that MXS stimulates whole cell Cl currents, whereas minoxidil (MX) stimulates K currents in dissociated eccrine clear cells. Using whole cell clamp techniques we observed that: 1) 1 mM MXS stimulated sweat secretion in vitro; 2) MXS depolarized the membrane potential by as much as 40 mV; 3) MXS stimulated membrane conductance, increased inward current and shifted the reversal potential to the right when physiological electrolyte solutions were used; 4) in symmetrical Cl (Cl/Cl) solutions without permeable cations, MXS induced outwardly rectifying current-voltage (I-V) relationships; 5) in the Cl/Cl solutions, the MXS-induced current responses to imposed voltage pulses showed time-dependent activation, especially at the depolarizing potentials; 6) the reversal potential of the MXS-stimulated I-V curves in the Cl/Cl solutions shifted to the right by 55 mV when [Cl] in the bath was decreased from 157 to 7 mM; 7) MXS did not elevate cytosolic Ca or cAMP, although prolonged exposure to a Ca-free solution abolished the effect of MXS and 8) MXS-stimulated conductance was partially inhibited by diphenylamine-2-carboxylic acid, a blocker of Cl channels. The data suggest that MXS stimulates Cl channels, most likely depolarization-activated, outwardly rectifying channels. In contrast, the parent compound MX hyperpolarized the membrane potential and stimulated outward current without elevating cytosolic Ca and was independent of extracellular Ca, suggesting that MX stimulates Ca-insensitive K currents.     

Scanning ion conductance microscopy of living cells

Currently there is a great interest in using scanning probe microscopy to study living cells. However, in most cases the contact the probe makes with the soft surface of the cell deforms or damages it. Here we report a scanning ion conductance microscope specially developed for imaging living cells. A key feature of the instrument is its scanning algorithm, which maintains the working distance between the probe and the sample such that they do not make direct physical contact with each other. Numerical simulation of the probe/sample interaction, which closely matches the experimental observations, provides the optimum working distance. The microscope scans highly convoluted surface structures without damaging them and reveals the true topography of cell surfaces. The images resemble those produced by scanning electron microscopy, with the significant difference that the cells remain viable and active. The instrument can monitor small-scale dynamics of cell surfaces as well as whole-cell movement.