CFTR REVIEW PAGE
ION THEORY: Imagine yourself a college student in the 1850s. And if you had asked your professor what exactly happens when a common salt such as sodium chloride is dissolved in water, he would probably have answered that the salt remains in the water as intact sodium chloride; unless you were to add electricity, where upon the sodium would dissociate into solid sodium and the chlorine as chlorine gas. But if you had asked the same question in the late 1880s you might have gotten the answer we now know today to be the more correct one, due in large part to work by the chemist Svante Arrhenius: that as soon as you add the salt to water, it dissociates (breaks up) because of the water into individual positively charged sodium ions and negatively charged chloride ions. Arrhenius came upon this belief while trying to accurately explain increases in conductivities that were seen of aqueous solutions when various salts, or "electrolytes", were added. The word ion comes from the Greek for "wanderer", because, when placed in an electric field, ions are seen to move in accord with the strength and direction of the electric field applied.
THE CELL MEMBRANE: Early cell biologists had begun to suspect that the envelope surrounding the cell was made out of lipid (fat) in part because of the work by Ernst Overton, in 1899. He showed that certain types of small uncharged dye molecules, which resembled lipids chemically, could make their way inside cells easier than other molecules which didn't look like lipids at all. Thanks to the work of Chevural in the early 1800s, the chemical and physical nature of fatty acids was understood reasonably well. Using strong bases and triglycerides as reagents, he had been able to work out the properties of these molecules now known to be involved in both energy storage and cell membrane production.
In 1925, a landmark experiment was performed by E. Gorter and F. Grendel, which involved taking red blood cells and dissolving the cell membranes in organic solvents. Since lipids "look" more like organic solvents, chemically, than they do water they can be purified away from the rest of the cell in this fashion. Once they had the purified lipid constituents of the cell membranes, they placed them on the surface of water. As predicted, the relatively small polar end of the lipid molecules associated exclusively with the surface of the water while the longer nonpolar parts of the molecules stuck up above the surface into the air. But the part of the experiment with historical significance came when they later squeezed the edges of the lipid film together using wooden floats. Gorter and Grendel were able to cause this surface film of lipid to double up on itself and form a two-layered (bilayer) membrane just like what we now know to be the true nature of nearly all cell membranes. This was the first experimental confirmation for the lipid bilayer.
ION CHANNEL HISTORY (con't): By the late 1800s, the chemical mechanism underlying nerve and muscle tissue messaging was still a mystery. It's somewhat remarkable that Ludimar Hermann, one of du Bois-Reymond's students was able to conclude during this period that nerve and muscle cells were capable of exhibiting a "self-propagating wave of negative charge which advances in steps along the tissue", since the mechanism of this phenomena remained elusive. Into this void of understanding, came several established physicists from the "German School" who were intent on providing a physical and chemical explanation for all of life's processes. One of these was Julius Bernstein. He made the first real theoretical contribution, for he postulated the ionic theory, the Nernst equation, and the assumption of a semi-permeable membrane surrounding nerve and muscle cells could all help explain the mounting electrophysiological evidence of the past century. His position was that differences in potassium ion (K+) concentrations on the inside and outside of the cell could account for the presence of the by then well-established phenomena of membrane potential ( i.e. membrane voltage). Sidney Ringer used a solution of water and ran it thru the vessels of an isolated heart from a frog in the 1880s and discovered that in order for the heart to continue beating salts needed to be present in the water. Specifically, sodium, calcium, and potassium salts were needed and they had to be in special concentrations relative to each other.
There was now a reasonable explanation for Matteucci's observation in the 1830s that dissected frog muscle tissue later exposed to the toxin that causes tetanus ("lock jaw") had a greatly reduced ability to contract (and therefore gave less electrical discharge). The tetanus toxin was somehow influencing the flow of ions in and out of these muscle cells. What was still lacking was a sound mathematical explanation of how and why ions could move in and out of muscle and nerve tissue. Another problem was that frog and mammalian neurons have very small diameters compared to some marine animals. If one was ever to measure the membrane potential on the inside of a cell, the electrode needed to be extremely small and the nerve cell relatively larger in diameter than what was then available.
British physiologist John N. Langley, in 1907, put forward the concept of "receptor molecules" on the surfaces of nerve and muscle tissue in an attempt to explain the direct effects of certain chemicals such as tetanus toxin on them.
In 1937, one of the first to make use of squid neurons to study ion currents was John Z. Young . Squid neurons are convenient because they are approximately 100 to 1000 times larger in diameter than other animal neurons. The ease of working with large neurons made important experiments possible for the first time, including the first intracellular recordings of the nerve cell action potential as well as the first measurements of the underlying ionic currents that produce them. Using squid neurons, it was also possible to remove the cytoplasm from inside them and compare the axon's intracellular ionic composition directly with the exterior of the cell (the axon is simply the extended portion of the neuron). This experimental arrangement made possible the invention in 1949 by Ling and Gerard of the glass microelectrode. With it workers could now study more directly and quantitatively the way in which ionic sodium and potassium concentration gradients could give rise to changes in membrane potential. These small intracellular electrodes were fashioned from glass capillary tubes which were heated and then stretched out until their diameters were less than a micron, much smaller than a neuron. The electrodes were then filled with a salt solution in order to detect changes in current once placed inside the cell by simply impaling the cell membrane with them.
Not long after this, using Squid neurons, Hodgkin and Katz removed sodium ions from outside the neuron and were able to conclude from their data on membrane potentials that sodium ions were what was responsible for the formation of the action potential. They reasoned this because, when sodium was not present, there was essentially no change in membrane potential (action potential) upon stimulation with a current. In essence, they found a simple linear relationship between the amount of sodium outside the neuron and the amplitude (strength) of the action potential. Less sodium gave less amplitude and vice versa. It was now ninety years after Helmholtz had first measured the speed of the action potential along the nerve of a frog. It was now clear that the way in which neurons were able to conduct messages along their length was by letting sodium in and potassium out of the cell at strategic times and in such a way as to generate a traveling message, in this case a traveling change in the membrane potential which can then be detected by another cell further down the line. By 1940 it remained to be determined the exact mechanism of how sodium and potassium ions flowed through the cell membrane. And if it turned out that they use specific "ion channels" for their movement, were they the same channels? Or were they separate channels specific for each type of ion?
The next improvement in instrumentation took place in the late 1940s by Kenneth Cole. This involved placing a second glass electrode inside the cell in order to "voltage clamp" the interior of the cell. Voltage clamping made it possible to keep constant the membrane potential on the interior of the cell even during the sodium influx during the action potential. Researchers could now distinguish the voltage effects caused by influx of sodium or efflux of potassium from changes those made deliberately by the experimenter. Until this improvement was made, it remained impossible to obtain enough resolution to conclude conclusively that the effect studied since Galvani's time in the frog leg was due to the presence of discrete ion channels which were imbedded in cell membranes of neurons and muscle cells.
The 1940s also saw the explanation by Hodgkin, Huxley, and Katz of the resting potential and action potentials in terms of movement of specific ions- K+, Na+, Cl- thru pores in axonal membranes. Next, Katz, Fatt, and Exxles were able to show that ion channels were also fundamental to signal transmission across the synapses. In this case, however, the channels would turn out to be regulated by ligands like acetylcholine, rather than changes in membrane votages. Later in the 1960s and 70s, many other small molecules and peptides would be discovered to help gate these channels, including glutamate, GABA, glycine, serotonin, dopamine, and norepinephrine. So far there are approximately 100. Another type of synapse totally electrical in nature would also be discovered by Furshpan in 1957. Here, gap junctions function to bridge electrical synapses and allow current to flow directly between neurons.
The time was now right for revisiting the effect seen by Matteucci on muscle tissue when he added the toxin that causes lock jaw (tetanus). One of the most informative toxins characterized by the squid axon/voltage clamp method in the 1960s was tetrodotoxin, an alkaloid neurotoxin isolated from fugu puffer fish. Tetrodotoxin was found to exclusively block sodium but not potassium flow thru the membrane and this provided strong evidence for the existence of specific sodium channels. Soon afterwards, the synthetic ion tetramethylammonium was shown to also specifically block ion flow, this time only potassium ions. This proved that the sodium and potassium channels were distinct channels and could be controlled differently. Hodgkin and Huxley were now in a position to develop the first mathematical theory to accurately describe the existence of ion channels. By 1955, radioactive tracer ions like potassium were being used to track movements of potassium across nerve axons. The explanation given for the observed results indicated that the ions were passing single file thru a "narrow pore", or channel.
In the early 1970s, "noise" seen in experiments on synapses between neurons and muscles were interpreted to be "openings" and "closings" of individual ion channels as they repeatedly bound and unbound acetylcholine. Also about this time purified antibiotic gramicidin ion channels were added to artificial lipid bilayers by Hladky and Haydon. They saw "steps" in increased conductance which they interpreted to be increases in ion conductances as the individual ion channels bound to the bilayers one at a time and started letting ions thru. In 1971, Hille, while studying the voltage-gated sodium ion channel from frog neuron, measured the various mobilities of several types of ions other than sodium to gain information on the structure and mechanism of ion passage thru an ion channel. He concluded that the part of the pore which acts as a "filter" and controls which ion moves thru it is about 3 by 5 Angstroms in size. This turns out to be about the same size as a sodium ion sitting next to a water molecule with two more water molecules on either side. He went on to show, using molecular models, that there must exist certain types of relatively weak non-covalent bonds (Hydrogen-bonds) between the ion and the channel itself in order to allow for efficient passage thru the channel. Also in the 1970s, Miller developed a method which enabled insertion of membrane vesicles or reconstituted proteoliposomes into pure membrane bilayers. In the 1990s, researchers would use a version of this technique to show once and for all that CFTR is indeed a chloride ion channel, among other things.
Until the 1970s, Hodgkin, Huxley and others had only been able to study ion channels in neurons collectively. The signals they measured were called "macroscopic currents" because there was simply no experimental method which could be used to isolate and characterize individual ion channels. In 1976, Erwin Neher and Bert Sakmann invented the "patch clamp" method. It has since been referred to as the single most important development in ion channel research in the last half of the 20th Century because with it they and others were able to make direct observations about ion channels individually. Having the patch clamp meant that there was now a way to distinguish different types of channels from one another, and it was soon found that they tended to fall into one of there categories: Voltage-gated, extra cellular ligand gated, and intracellular ligand gated. And some were able to pass more than one type of ion through them. The patch clamp method makes it possible to detect currents which are smaller than 0.000000000001 Amps! Plenty sensitive enough to measure ion flow through single ion channel molecules. In 1970, there were a total of 3 scientific papers with the word "channel" in their titles. By 1980 over 300, and in 1990 over 1000.
The patch clamp was made possible in part because of improvements in amplifiers, as well as cell culturing techniques. Basically, a tiny glass pipette filled with salt solution is placed against a cell membrane where it is believed there is a single ion channel. If this is the case, then one can use the flow of ions in or out of the ion channel to detect the flow of ions as one would detect a current in a wire. Since ions carry a net charge, not unlike electrons in a wire conductor, it is possible to achieve extremely high resolution. The patch clamp was the first method to be developed for studying the activity of individual protein molecules (ion channels) and it remains one of the few methods today for doing this.
Patch Clamp Method (below): (A) current in the form of ions moving through an individual ion channel can be monitored with this technique. (B) Tracing (recording) of ion flow. Y-axis represents current, X-axis is time usually in msec. Plasma membrane (cell membrane), channel (ion channel),