Life at the Cell and Below-Cell Level. The Hidden History of a Fundamental Revolution in Biology
"Dr. Ling is one of the most inventive biochemist I have ever met."
The Living State: Electronic Mechanisms for its Maintenance and Control
This section begins with a bit of history. It shows how interaction with other scientists and their ideas provided the stimulus as well as conceptual stepping stones toward developing LFCH into a unifying physico-chemical theory of life called the association-induction hypothesis (AI Hypothesis, AIH). As such, it is the first in history.
The development of the AI Hypothesis took three steps: LFCH in 1952; the association-induction hypothesis (proper) in 1962; the subsidiary Polarized Multilayer (PM) theory of cell water in 1965. The late-arriving PM theory was presented before the AI Hypothesis proper because, like LFCH presented still earlier, the PM theory also deals with the static associative aspects of cell physiology. The AI Hypothesis proper, in contrast, deals also with the dynamic inductive aspects of cell physiology. Built upon the association of ions, water and proteins, obviously the AIH proper can only be presented last.
There is a second reason for the inverted order of presentation. The LFCH and the PM theory are focused largely on molecular events, be it ion adsorption or water polarization-orientation. The AI Hypothesis proper, in contrast, ventures out into the realm of electronic control mechanisms. Surprising as it may seem, this too is a direction that had not been pursued before—until the advent of the AI Hypothesis.
The presentation of the electronic control is divided into two parts. The first part presented in this chapter (Chapter 14) deals with the electronic control mechanism itself and its role in what is to be described as the living state. The second part to be presented in the succeeding chapter (Chapter 15) deals with electronic mechanisms underlying physiological activities of diverse kinds and their control. We are now at the beginning of the first part.
As pointed out earlier, I began my training as a cell physiologist under Prof. Ralph W. Gerard—a better teacher and mentor I could never have found, far-reaching in interests and knowledge, brilliant in thoughts and actions, yet unwaveringly nurturing to his students like myself. As a graduate student, I improved the procedures in making and filling with KC1 solution the capillary glass electrode so that one could use it to make accurate and reproducible measurements of the electric potentials of living cells. Thus armed, Prof. Gerard and I continued the study of the resting potentials of frog muscle cells that he and Judith Graham had begun earlier.88, 441, 442, 443 This line of research soon led me to the subject of selective K+ accumulation in living cells and the eventual proposal of what was later referred to as Ling's Fixed Charge Hypothesis (LFCH).
In presenting the LFCH in 1952 96 Fig 5 and in a short review written in 1955,145 I pointed out that in theory the same fixed charge system (i.e., β- and γ-carboxyl groups)—which selectively adsorb K+ over Na+ in the bulk-phase cytoplasm—could also act as the seat for the cellular resting potential when transplanted to the cell surface (Figure 4C).
Hodgkin and Katz made the epoch-making discovery in 1949 that during an action potential, the cell membrane switches its preferential permeability to K+ to a preferential permeability to Na+.233 In the light of this exciting new knowledge, I asked myself the following question: If the resting potential arises from the preferential adsorption of K+ on the fixed β- and γ-carboxyl groups at the resting cell surface—rather than from its preferential membrane permeability—could the same surface β- and γ-carboxyl groups switch their preference for K+ to a preference for Na+ during an action potential? If so, how? Clues to possible answers came from studies of the ion exchange resins and of the glass electrodes.
In support of LFCH, I cited in 1952 various inanimate fixed-charge-systems. Included are permutits, soils and synthetic ion exchange resins.96 p 773 They all carry fixed anionic sites and they all selectively accumulate K+ over Na+. One recalls that long before, Benjamin Moore and Herbert Roaf had already quoted soils in a similar context77 (Chapter 7).
Ion exchange resins are better models than soils. First, ion exchange resins are simpler in structure and thus easier to understand. Second, it is the product of a rapidly advancing new technology. Indeed, even as the LFCH was trying out its new wings, so to speak; more and more was being discovered on the relationships between the chemical makeup of an ion exchange resin and its ion-exchange properties.15 pp 261-263; 478 Thus the K+-selecting cation exchange resins I referred to in 195296 all carry fixed anionic sulfonate groups. When ion exchange resins carrying fixed carboxyl groups became available, they were found to select Na+ over K+ (143)—a new finding first brought to my attention by Prof. E. J. Conway of Dublin in his argument against my thesis that (β- and γ-) carboxyl groups in living cells selectively adsorb K+ over Na+ [10.1(3)]. At first, I was dismayed by this "attack." As time wore on, I began to appreciate it more and more for the underlaying truth it called to my attention.
At that time I already strongly believed that β- and γ-carboxyl groups in muscle cells selectively adsorb K+ over Na+—for among other reasons, no other fixed anionic groups in the living cells are numerous enough to provide enough adsorbing sites for all the cell K+. Yet carboxyl groups in ion exchange resins do not selectively adsorb K+ over Na+ as my theory demands, but do just the opposite.143 Could one kind of carboxyl group act one way and another kind act another way? If so, how?
J. I. Bregman, who reviewed the subject of cation exchange resins in 1953,143 p 135 brought attention to the fact that the sulfonate group (which selects K+ over Na+ is strongly acidic and has a low pK value, but the carboxyl group (which selects Na+ over K+) is weakly acidic and has a high pK value. For explanation of the reversal of ion selectively, however, he cited Teunissen and Bungenberg de Jong,319 who attributed the difference in the rank order of preference for K+ and Na+ in various colloids to the different polarizability of the sulfonate and carboxyl groups they carry respectively.
Digging deeper (much later), I discovered that Bungenberg de Jong and his coworkers had done a great deal of important work on the subject of ion selectivity in colloids—published mostly in the 1930's and 1940's. To avoid misrepresenting their ideas, I cite below their opinions mostly verbatim.
H.G. Bungenberg de Jong—the same colloid chemist cited earlier—Teunissen and others studied the migration of colloids in water under a DC electric field. They noted that in response to the introduction of different cations in the bathing solution, the direction of migration of the colloids sometimes changed. Bungenberg de Jong (and coworkers) attributed this directional change in migration to a change in the net electric charges of the colloid, and called it "charge reversal."61 pp 159-334 Bungenberg de Jong further pointed out that "If ...the reversal of charge is generally caused by fixation of a sufficient amount of (positively charged) cations on the ionized (negatively charged) groups (of the colloids—then the affinity of cations and ionized groups must depend on valency, radius and polarizing power of the cation and on the polarizability of the negatively charged ionized groups of the colloid" (ital. Bungenberg de Jong's).61 p 287
He continued: "If we consider for instance the monovalent ions, Li, Na and K, then—exclusively from the point of view of "field strength"—on the surface of these ions, the fixation on a given negatively charged ionized group will be easiest in the case of the smallest ion—Li—and will be increasingly more difficult for the larger Na and Ê ions. But as a second influence we must consider the energy of polarisation. If the ionized group is more polarisable than water, then the polarisation energy is added to the Coulomb energy. In this case, the above order of cations will not be disturbed, on the contrary, the spread of Li>Na>K will possibly be strengthened.... In the case that the polarisability of the ionized group is smaller than that of water, then the polarisation energy of the water molecules (cation hydration) will oppose the Coulomb energy. Now, the Li ion, the most hydrated ion, will have the greatest tendency to rest in the perfect hydrated state free in the medium." 61 pp 287-288 (And the selectivity order will be reversed to K>Na>Li, added by GL.)
Bungenberg de Jong and his coworkers also attempted to explain the ion-selectivity-order differences among colloids carrying similar carboxyl groups also in terms of their dissimilar polarizabilities. The following direct quotation clarifies what they were thinking.
"It seems quite natural to ascribe the lesser polarisabilities of the carboxyl groups of the latter (arabinate, the carboxyl group of which was compared with the carboxyl groups of oleate—added by GL) to a certain constitutional influence. They (arabinates) are derived from polymer carbohydrates and thus the presence of hydroxyl groups in the neighbourhood of the carboxyl groups could be the cause of the decreased polarisability..."61 p 293
To test the theory that it is the anionic-group polarizability that plays the key role, Teunissen, Rosenthal and Zaayer144 showed in 1938 that the possession of many hydroxyl groups of arabinate is correlated with a selection sequence of K+ > Na+ > Li+ and Mg++ > Ca++ > Sr++ > Ba++ while oleate devoid of hydroxyl groups shows inverted sequences.
In 1955, I presented my new theory of cellular electric potential at the Federation Meetings at Atlantic City. In the verbal presentation as well as the printed abstract, I pointed out that my new theory of cellular electric potential is "closely related to that of the glass electrode potential."145
In my audience was Prof. Harry Grundfest of the Columbia University. Also attending the meetings (but not my talk) was a young MD from Harvard, George Eisenman. Eisenman, Donald Rudin and Jim Casby had just been invited to set up a Basic Research Department in the newly-founded Eastern Pennsylvania Psychiatric Institute (EPPI) in Philadelphia. Among their initial research interests was the electrical activities of the central nervous system. In the course of this study, they found it necessary to determine the Na+ concentration in the (spinal) fluid in the (narrow) central canal of the spinal cord of an experimental animal. At that time, Na+ selective microelectrodes required to do this job were not commercially available. So they purchased a high-powered electric furnace and set out to make their own Na+ sensitive electrodes. As Eisenman and coworkers varied the chemical composition of the glass electrodes, electrodes with different selectivity sequences among the five alkali-metal ions, Li+, Na+, K+, Rb+ and Cs+ were created.
Grundfest told Eisenman what I had presented earlier at the Federation Meeting and how I was studying the glass-electrode as a model for the electric potentials of living cells. Next thing you know, I was invited to visit the Department of Basic Research at EPPI. And during the visit I described to Eisenman, Rudin and Casby my theory of enhanced counterion association with charge fixation and that of selective K+ adsorption on the basis of what, in simpler terms, may be described as differences in the field strength experienced by K+ and by Na+ from the fixed carboxyl groups.
Later, over a lunch at a nearby Howard Johnson Restaurant on Henry Avenue, I also thought aloud with my new friends as to how my 1952 model could be modified to fit some of their new findings of varying ion-selectivity order. I recall suggesting a combination of my model of fixed anions with the concept of rigid pores on the glass surface. And how the rigid glass wall of a narrow pore containing a fixed anion at its bottom may force the more hydrated ions to lose some of their water of hydration, thereby creating a different rank order of selectivity. But it was just a passing thought and not further pursued by me. (At that time, I was not aware of Bungenberg de Jong and his coworkers work cited above. I learned of it later.)
On the basis of these premises, they further postulated that among the five alkali-metal ions, the least hydrated Cs+ would be the first to shed its hydration shell, Rb+, the next to do so and so on in an orderly fashion. In consequence, eleven orders of selective ion adsorption were predicted.146 They then showed that the diverse rank order of ion selectivity they observed in the experimental glass electrodes they made agree with these orders.148 pp 274-279
In line with our newly-developing converging interests, Eisenman urged me to leave my (tenured) associate professorship at the Neuropsychiatric Institute of the Medical School of the University of Illinois and join him as a (tenured) Senior Research Scientist at EPPI. He was a persuasive salesman and the advantages offered were substantial. I accepted his offer with no hesitation. On March 1, 1957 my wife Shirley, our baby son Mark and I arrived at Philadelphia, where forsythia was just beginning to bloom.