The Ca(2+) channel alpha(1S) subunit (Ca(V)1.1) is the voltage sensor in skeletal muscle excitation-contraction (EC) coupling. Upon membrane depolarization, this sensor rapidly triggers Ca(2+) release from internal stores and conducts a slowly activating Ca(2+) current. However, this Ca(2+) current is not essential for skeletal muscle EC coupling. Here, we identified a Ca(V)1.1 splice variant with greatly distinct current properties. The variant of the CACNA1S gene lacking exon 29 was expressed at low levels in differentiated human and mouse muscle, and up to 80% in myotubes. To test its biophysical properties, we deleted exon 29 in a green fluorescent protein (GFP)-tagged alpha(1S) subunit and expressed it in dysgenic (alpha(1S)-null) myotubes. GFP-alpha(1S)Delta 29 was correctly targeted into triads and supported skeletal muscle EC coupling. However, the Ca(2+) currents through GFP-alpha(1S)Delta 29 showed a 30-mV left-shifted voltage dependence of activation and a substantially increased open probability, giving rise to an eightfold increased current density. This robust Ca(2+) influx contributed substantially to the depolarization-induced Ca(2+) transient that triggers contraction. Moreover, deletion of exon 29 accelerated current kinetics independent of the auxiliary alpha(2)delta-1 subunit. Thus, characterizing the Ca(V)1.1 Delta 29 splice variant revealed the structural bases underlying the specific gating properties of skeletal muscle Ca(2+) channels, and it suggests the existence of a distinct mode of EC coupling in developing muscle.

In normal airway epithelium, the cystic fibrosis transmembrane conductance regulator (CFTR) transports Cl^- ions to the apical surface of the epithelium paralleled by the flow of water through transcellular and paracellular pathways. The hypothesis was tested whether CFTR not only regulates the transcellular but also the paracellular shunt pathway. Therefore, we performed measurements of transepithelial electrical resistance (TER) and paracellular ¹⁴C-mannitol permeability in wtCFTR (16HBE14o^-) and delF508-CFTR (CFBE41o^-) expressing human bronchial epithelial cells. Under resting conditions, CFBE41o^- cell monolayers exhibit a higher paracellular permeability and lower TER as compared to 16HBE14o^- monolayers. Stimulation of CFTR by cAMP induces opposite effects in the two cell lines. 16HBE14o^- monolayers show a sharp decrease of TER, in parallel with a concomitant increase of paracellular permeability. The change in paracellular permeability is mediated by a myosin II dependent mechanism because it can be blocked by the myosin light chain kinase inhibitor ML-7. In contrast, CFBE41o^- cells respond to cAMP stimulation with a decrease of paracellular permeability, paralleled by slight increase of TER. We conclude that stimulation of wtCFTR increases vectorial transcellular salt transport and, simultaneously, the paracellular permeability allowing water to follow through the paracellular pathway. In contrast, in CF epithelium cAMP stimulation increases neither vectorial salt transport nor paracellular permeability which is likely to contribute to the CF pulmonary phenotype. Taken together, our results link CFTR dysfunction to an improper regulation of the paracellular transport route.

It has been reported recently that the cystic fibrosis transmembrane conductance regulator (CFTR) besides transcellular chloride transport, also controls the paracellular permeability of bronchial epithelium. The aim of this study was to test whether overexpressing wtCFTR solely regulates paracellular permeability of cell monolayers. To answer this question we used a CFBE41o– cell line transfected with wtCFTR or mutant F508del-CFTR and compered them with parental line and healthy 16HBE14o– cells. Transepithelial electrical resistance (TER) and paracellular fluorescein flux were measured under control and CFTR-stimulating conditions. CFTR stimulation significant decreased TER in 16HBE14o– and also in CFBE41o– cells transfected with wtCFTR. In contrast, TER increased upon stimulation in CFBE41o– cells and CFBE41o– cells transfected with F508del-CFTR. Under non-stimulated conditions, all four cell lines had similar paracellular fluorescein flux. Stimulation increased only the paracellular permeability of the 16HBE14o– cell monolayers. We observed that 16HBE14o– cells were significantly smaller and showed a different structure of cell-cell contacts than CFBE41o– and its overexpressing clones. Consequently, 16HBE14o– cells have about 80% more cell-cell contacts through which electrical current and solutes can leak. Also tight junction protein composition is different in ‘healthy’ 16HBE14o– cells compared to ‘cystic fibrosis’ CFBE41o– cells. We found that claudin-3 expression was considerably stronger in 16HBE14o– cells than in the three CFBE41o– cell clones and thus independent of the presence of functional CFTR. Together, CFBE41o– cell line transfection with wtCFTR modifies transcellular conductance, but not the paracellular permeability. We conclude that CFTR overexpression is not sufficient to fully reconstitute transport in CF bronchial epithelium. Hence, it is not recommended to use those cell lines to study CFTR-dependent epithelial transport.

We recently discovered that hyaluronan was exported from fibroblasts by MRP5 and from epithelial cells by cystic fibrosis (CF) transmembrane conductance regulator (CFTR) that was known as a chloride channel. On this basis we developed membrane permeable analogs of hyaluronan disaccharide as new class of compounds to modify their efflux. We found substances that activated hyaluronan export from human breast cancer cells. The most active compound 2-(2-acetamido-3,5-dihydroxyphenoxy)-5-aminobenzoic acid (Hylout4) was tested for its influence on the activity of epithelial cells. It activated the ion efflux by normal and defective ΔF508-CFTR. It also enhanced the plasma membrane concentration of the ΔF508-CFTR protein and reduced the transepithelial resistance of epithelial cells. In human trials of healthy persons, it caused an opening of CFTR in the nasal epithelium. Thus compound Hylout4 is a corrector that recovered ΔF508-CFTR from intracellular degradation and activated its export function.

No abstract