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It has been known for quite some time that CFTR is a cAMP regulated chloride ion channel whose mutation is intimately linked to cystic fibrosis (CF). However, the abnormality in the chloride ion channels is not the only biochemical peculiarity associated with the disease. Of particular interest to researchers is the finding that sodium ion (Na+) absorption in the cells of CF patients is both elevated and positively related to the cAMP concentration, whereas, in normal cells, cAMP concentration is inversely related to the rate of Na+ absorption. The malfunction of the Na+ pump becomes immediately apparent, for the primary symptom of CF is a chronic bacterial infection of the lungs, due to the abnormally low viscosity of the fluid which bathes the cellular epithelium; by pumping ions into the airway, rather than into the cell, a thermodynamically unfavorable concentration gradient of sodium ions is is established, in which the airway is hypotonic and osmosis is kinetically favored. In the case of CF, the local chloride pump is inoperative, and the sodium pumps are incredibly overactive, importing Na+ from the airway, and taking water out of the fluid inside the lungs in the process. Therefore, is is inappropriate to place the sole responsibility for the low fluid viscosity directly upon the mutation of the CFTR protein. Studies have demonstrated that the elevated Na+ absorption is not due to an overexpression of the Na+ transport protein, ENaC, in the membrane, nor can cAMP sensitivity be linked to a genetically mutated ENaC. The possibility that the altered CFTR indirectly led to the Na+ channel phenomena by altering the functionality of normal ENaC formed the basis of a hypothesis which was tested by M. Stutts, C. Canessa, J.Olsen, M. Hamrick, J. Cohn, B. Rossler, and R. Boucher (Science, v. 269, August 11, 1995) in the experiment explained here.
In order to formulate a system of experiments which would be comprehensive to the study, the researchers first pooled all the knowledge available which was relevant to their topic. In addition to what was outlined above, they also noted that the Na+ absorption, via ENaC, was inhibited by the the presence of amiloride and its analogs, and that previous studies demonstrated that the same process was stimulated by the presence of forskolin in the CF cells. Furthermore, the three subunits which compose ENaC, alpha, beta, and gamma, function almost identically to the subunits of a similar transport protein found in rats, rENaC. Armed with this knowledge and considering the available techniques of experimentation, the scientists devised a stratagem which would determine whether or not the presence of CFTR in epithelial cells was responsible for regulating ENaC's function in the cell. Therefore, the researchers' ultimate goal was to study the behavior of ENaC in both the absence and presence of CFTR.
The first task at hand was to define a standard of measurement with which to compare the experimental cells: a control. Since the ion concentration in cells is controlled by a series of membrane proteins, the simplest standard for the researchers to measure would be the cellular ion current. An unequal exchange of ions causes the cell membrane to reach a higher level of energy because the molecules of the membrane are more excited than their normal energy level. When a system falls towards equilibrium, the excess energy in the system is released; the energy release in a cell is characterized by a decreasing level of membrane potential (Isc). Conversely, an increasing membrane potential denotes a higher level of energy, or, in other words, more ions flowing across the cellular membrane. The hydrolysis of ATP, which requires active transport, is constantly occurring, thus a living cell always has a characteristic charge. The rate at which a current flows across the membrane, or the membrane potential, can be measured in micro-amperes per cm2. These units of measure (mV and A/cm2) are based on the principle of active transport, and serve as a scale upon which the differences between cells, resulting from varying conditions, may be qualitatively observed.
The next challenge to be tackled was how to relate the property of current generation to a physical system while allowing ease and accuracy in measurement. The scientists chose to utilize a control technique which accomplished both of these desires, and also could mimic the chemistry of human epithelial tissue: transfection of the desired genes for ion function into controlled, functional cells. Clones of Maden Darby Canine Kidney (MDCK) cells, naturally expressing few ENaC and CFTR, were cotransfected with complementary DNA (cDNA) strands which coded for the three subunits of the rENaC protein. During the 24 hour induction time, the cell produced mRNA corresponding to the transfected DNA and translated the mRNA into the correct amino acid sequence, allowing the membranes to express the desired protein. To insure that this process had occurred in the transfected cells, they were metabolically labeled with [35-S] methionine and then were exposed to and precipitated by antibodies corresponding to the alpha, beta, and gamma subunits of rENaC, in a process known as immunopricipitation. The protein subunits were then separated using 10% resolving SDS-polyacrylamide gel electrophoresis and assayed to give Western Blot results which corresponded to results standard for the rENaC subunits. An identical test was run on the control cells which revealed, as expected, that little ENaC had existed prior to transfection. Furthermore, the non-induced transfected cells exhibited a similar lack of expression.
Determining the functionality of the transfected MDCK cells was the next step in creating a control. To demonstrate the effect of rENaC on transfected and induced cells, the currents of the differentiated cells (parent non-induced and induced, and rENaC transfected induced and non-induced) needed to be measured. Therefore, these differentiated cells were grown on separate collagen supports which could then act as a selectively permeable barrier, whose properties were determined by the cell-type compositions. By altering solute concentrations on either side of the collagen barrier, permeability of the cells to ions can be measured under varying conditions using current electrodes. The results of this test are shown in the graph. It should be noted that only the cells which had been both transfected and induced were capable of generating a substantial current. Having established a method to create a functional control, the researchers went on to discover the effects of [Cl-] on rENaC. As the same graph demonstrates, [Cl-] has little effect upon the current, therefore the measured current can be attributed to Na+. Next, the effects of amiloride and several of its analogs were observed by measuring the change in current associated with the shifting inhibitor concentrations (see graph). The experimental results, according to the graph, closely parallel the affects found in the human airway.
Now that the behavior of rENaC had been characterized, the effects of CFTR on the Na+ pump could be explored. This test was conducted by exposing the MDCK/rENaC cell layers to Ad5-CBCFTR adenovirus, which contains cDNA encoding for human CFTR. As with MDCK/rENaC experiment, a Western blot was run to ensure that the CFTR protein had been expressed. Also, Isc was measured across the layer in a Cl- free environment, in order to gain an accurate measurement of only Na+ channel activity (see graph). After induction, the transfected CFTR decreases sodium current by approximately 28%. This finding was momentous, for it gave credence to the hypothesis that the expression of CFTR in cells containing rENaC has had a profound effect on rENaC's functionality. (It would, however, have been more conclusive if the relative amounts of each protein would have been determined and compared to a "normal" epithelia cell, to ensure that the difference was not a result of concentration dependent interference.) Additional conformation of this hypothesis was rendered by a comparison of the forskolin induced changes between rENaC and rENaC+CFTR induced cultures. Though the affects of amiloride and the Na+ current are generally independent of the presence of CFTR, forskolin functions as an activator in the absence of CFTR and an inhibitor in its presence. These characteristics are displayed in the graph. The fact that CFTR can also significantly alter the consequences of the effectors was a further indication that this protein may have an indirect role in CF. Another interesting observation was that in the presence of forskolin, amiloride acts as a very weak Na+ current activator, regardless of CFTR presence (see graph).
The hope of proving their hypothesis true inspired the researchers to run one last series of experiments in order to ensure a greater degree of certainty in their conclusion. They recognized the fact that the rate of transepithelial sodium ion currents were limited by the Na+ permeability of the membrane (not the Na+/K+ ATPase pump). So, they sought to examine regulatory interaction at the cellular level. Until this point, layers of cells had been used to obtain results; now, the precision desired required the use of a method capable of testing effector regulation on a much smaller scale. This requirement was satisfied by the whole-cell voltage clamp, a technique which allows the solute concentrations of the interior of the cell, in addition to the exterior, to be regulated. Continuity between a source of a controlled solution and the interior of a single cell is accomplished by allowing a selected cell's bilayer to adhere to the end of a small pipette, via the hydrophobic effect. The portion of the membrane covering the mouth of the pipette can be removed by applying slightly unequal fluid pressure, effectively making the interior of both the cell and the pipette continuous, so that whatever is added to the pipette diffuses into the cell. A small electrode, attached to an adjustable voltage meter, is inserted into the pipette, so that the membrane potential can be controlled.
Again, to isolate the significant variables, dysfunctional cells, 3T3 fibroblasts, were transfected with either rENaC or both rENaC+CFTR, then they were induced. The success of these two phases was demonstrated by immunoblot and immunofluorescence comparisons, as well as the fact that the parent inward whole-cell currents were minor, relative to the current of the induced fibroblasts. Further tests generated the data represented in the following graphs. (Note: the significance of the first pair of graphs in each set of the current vs. clamp voltage charts can be summarized as follows: the slope of the functions (change in current/change in voltage) is directly related to activation. The graphs in set A show that amiloride has no effect on parent fibroblasts, but has negative effect on fibroblasts treated with rENaC. Graphs in set Bdemonstrate that in the absence of CFTR, the rENaC responds positively to the increased levels of cpt-cAMP, whereas the inverse is true in the presence of CFTR. Another graph indicates that the 3T3-rENaC+cpt-cAMP protein complex is stimulated by the addition of amiloride, while the same addition results in inhibition of the 3T3-rENaC+cpt-cAMP protein complex. In other words, it was proved that amiloride treatment really did negate all of the cpt-cAMP induced changes.
These results confirm the finding that the sodium currents generate by rENaC are fundamentally altered by the presence of functional CFTR. As a cAMP dependent, negative regulator of Na+ channels, as well as a cAMP regulated Cl- channel, CFTR controls the ion concentrations within the airway (see graph), and, consequently, the viscosity of the fluid in that region. Thus, if a functional protein causes a relatively high osmotic potential in the lungs, it would be logical to suggest that a dysfunctional variety of cell, such as the CF mutant, would lead to lowered viscosity within the lungs, therefore creating the mucus which is the trademark of CF as a fatal condition.
