Cell Biology International ISSN 1065-6995 doi: 10.1002/cbin.10382
COMMENTARY
Cell electrical properties: reconsidering the origin of the electrical potential Gerald H. Pollack* Department of Bioengineering, University of Washington, Seattle 98195, WA, USA
Introduction The interior of the cell commonly has a negative electrical potential of 50–100 mV, a fact known for more than a half century. To create this potential difference, the ionic contents inside the cell must differ from those outside the cell. I present evidence that the potential difference arises, at least in part, from the unique characteristics of intracellular water, rather than from the action of membrane transporters (pumps) and channels.
Pump and channel issues Textbooks continue to espouse the long-held view that certain ions (and other substances) get pumped into or out of the cell, and subsequently re-enter and exit through selective channels. These membrane-based processes are widely presumed to create intracellular negativity. Pump and channel mechanisms have become so deeply ingrained into modern cell biology as to be essentially axiomatic. A number of criticisms lodged against those mechanisms have seldom been seriously addressed. One of them has focused on membrane pumps. Pumps require energy, and Ling (1997) showed that the cell could not possibly generate the energy required to power even the sodium pump, let alone the many other membrane pumps that have since been claimed to exist. Ling reviews the evidence on his website www.GilbertLing.org and argues that there has been no meaningful response to this proposal. Energy issues notwithstanding, the broad scientific community, in embracing the pump concepts, are confident of its explanatory power and consequent adequacy. However, the above-mentioned challenge remains unaddressed, and until it is, some doubt must linger. Membrane channels occupy a similar place in contemporary thinking, with a huge body of evidence seeming to underlie the existence of selective channels, their number now exceeding 400, with each channel being selective for a
different ion or solute. Therapeutic pharmaceutical agents commonly are claimed to act on one or other of these channels, thereby producing a physiological response. Like pump mechanisms, channels have become broadly accepted. Two challenges have yet to receive meaningful responses (Pollack, 2001). The first questions the original evidence for the existence of single ion-selective channels, which came from the classic paper by Neher et al. (1978) who used a patch of membrane. When the potential difference across that patch was raised beyond a certain threshold, pulse-like currents of consistent magnitude began flowing through the patch. Channel openings and closings were assumed to account for these discrete currents. However, similar pulselike currents appear in patches that have no channels, as has been corroborated by Sachs and Qin (1993), Lev et al. (1993) and Woodbury (1989). The original papers of Neher and Sackmann hinted at such difficulties, which were quickly dismissed. In an extensive series of experiments, Thomas Heimburg’s group at the Nils Bohr Institute more recently reported similar results (Laub et al., 2012); i.e., membranes with TRP (transient receptor potential) channels show conductance behavior indistinguishable from those of synthetic lipid bilayers, containing no channels at all. (A representative result is given in Figure 1). One would think this evidence to be troubling. As the presence of pulse-like currents of consistent amplitude constitutes the primary evidence for the existence of selective membrane channels, evidence of similar behavior in specimens with no channels suggests prudence. The second channel-related issue is conceptual. Suppose the membrane contains 400 channels, each selective for a particular solute or ion. When a particular channel opens, its respective solute passes through while other solutes remain excluded, and the channel then closes. This seems simple and elegant, but there is a problem. Solutes are of varied size and nature. Consider the channel that is selective for the largest of the 400 solutes. When that channel opens, what prevents the smaller solutes from passing through at the
Corresponding author: e-mail: [email protected]
Cell Biol Int 9999 (2014) 1–6 © 2014 International Federation for Cell Biology
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Figure 1 Patch clamp recording of a synthetic lipid membrane with no proteins or channels. Eight representative 2.5 s segments out of a 30-min recording. From Laub et al., 2002.
same time? If the channel is genuinely selective, it has to prevent all 399 smaller solutes from passing through. The 399th largest must prevent all 398 smaller ones from passing through, etc. It can be likened to the dog door, which can flap open to allow your pet dog to pass through, but it would not prevent a cat, ferret, and mouse from passing through at the same time. This theoretical conundrum has received at least some attention. In the classical work of MacKinnon and his colleagues (Zhou et al., 2001), the authors explained how a channel might distinguish between sodium and potassium ions, a finding important enough to have earned the senior author a Nobel Prize. It went partway towards dealing with the selectivity challenge; however, the treatment dealt principally with two ions of similar character. The selectivity problem is more extensive, for how could a channel large enough to pass a bulky molecule exclude a molecule one hundredth of its cross-section, such as water? If we take the fallback position and admit that the large channel does not really exclude all those smaller molecules, and that they may actually pass through, down their respective concentration gradients, why does the cell need channels devoted exclusively to those smaller molecules? If large channels can pass water, why does the cell need an aquaporin channel devoted exclusively to water, which seems like redundancy? The challenge of selectivity has to be addressed; at the very least, a convincing theory has to explain how a channel can permit passage of a particular 2
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solute, while excluding all solutes of smaller diameter, including some that are much smaller; otherwise the selective channel concept seems bereft of theoretical underpinning. In sum, it is thought that the cell’s negative electrical potential arises from the action of membrane pumps and channels. For most investigators these mechanisms seem acceptable and adequate, although still based on some assumptions and possibly shaky premises. However, the conceptual and experimental challenges raised above to the fundamental underpinning of these mechanisms needs to be fully addressed. For those who consider these issues minor compared to all the positive evidence, consider the conundrum of crowding. Membrane channels and pumps are continually being identified. The channel count may exceed 400, but the transporter count totals 1,000 (some colleagues put the “unofficial” transporter count at several thousand). To operate effectively, each of these entities must appear multiple times on the membrane of each cell. Even if some functions are shared, nevertheless, tens of thousands of pumps/channels must crowd into the cell membrane, along with the various receptors and other functional entities—it must be hugely crowded. The crowding problem grows more acute with time; if, as seems to be the case, the membrane’s surface area remains relatively fixed while additional channels/pumps appear, for example, for each newly synthesized drug, will membrane space eventually run out, unless existing channels are commandeered? These are not complicated arguments, but do they imply a logical conundrum. How certain we can be that the prevailing paradigm explaining the cell’s handling of ions is adequate? Can we be certain that the electrical potential of the cell is generated by the actions of pumps and channels?
Gel electrical potentials and cell electrical potentials The first inkling that the cell’s negative electrical potential might involve water came from a simple observation: the similarity of cells and gels. The cell cytoplasm is gel-like. Many cell biologists have routinely worked with “demembranated” cells. Following demembranation by mechanical or chemical means, the rest of the cell hangs together like Jell-O; it does not dissolve into the surrounding milieu as would an aqueous solution. The gel-like nature of the cytoplasm has long been established (Frey-Wyssling, 1953), and further evidence gives additional force to that consistency (Pollack, 2001). Electrical features of cells and gels are also similar; one can measure the gel’s electrical potential (more accurately the potential difference between inside and outside of the gel) by sticking a microelectrode in a gel. It is remarkable that the electrical potential of a gel can be similar to the electrical Cell Biol Int 9999 (2014) 1–6 © 2014 International Federation for Cell Biology
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potential of the cell: both can have negative potentials of the order of roughly 100 mV (Pollack, 2001; Zheng et al., 2006). Yet, the gel has no membrane, nor any channels or pumps. Either different mechanisms govern the gel’s and the cell’s potential or surface membranes have little or nothing to do with the cell’s electrical potential. We previously thought that both of these negative potentials might arise from the internal negatively charged proteins/polymers. The negativity of these solids might explain the observed negativity. This supposition must, at least partially, be valid. However, some difficulty arose when trying to relate this negativity to the rapid shifts of electrical potential occurring in excitable cells, as manifested in action potentials. The protein/polymer charge explanation seems inadequate; the problem is therefore more complicated than this alternative simple explanation indicates. Thus, we have to consider more carefully the role of water, by far the most populous molecule inside the cell. The idea of water’s contribution became compelling when it was discovered that water could bear negative charge. I will return to the relation between water’s negative charge and the cell’s negative potential after briefly explaining the unexpected finding of water’s negativity and some of its consequences.
A “New” phase of water? There is a fourth phase of water (Pollack, 2013; The Fourth Phase of Water: Beyond Solid, Liquid and Vapor; see also footnote), which is found next to water-loving (hydrophilic) surfaces such as proteins and other charged macromolecules. The phase is more extensive than originally conjectured, projecting out from surfaces by many molecular layers. It exists almost everywhere throughout nature, including the cell. Details of this newly identified phase of water have been described in many papers, and many basic findings have been compiled in the above-mentioned book, most of which require exploration in their nature and applications for technology. The book also deals with water’s many known anomalies, turning them into explained features. The existence of a fourth phase may seem unexpected; however, that should not be entirely so. A century ago, the respected physical chemist Sir William Hardy argued for the existence of a fourth phase; and a number of authors over the years have found evidence for some kind of “ordered” or “structured” phase of water. New
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experimental evidence not only confirms the existence of such an ordered, liquid-crystalline phase, but also details its remarkable properties. The energy for building this water structure comes ultimately from the sun. Radiant energy converts ordinary bulk water into ordered water, building this ordered zone inside and outside of biology. All wavelengths, ranging from UV through visible to infrared, suffice. Near-infrared and mid-infrared energy are the most capable (Chai et al., 2009). Water absorbs infrared energy freely from the environment and uses it to convert bulk water into liquid crystalline water covering hydrophilic surfaces of all types—which we call “exclusion zone” or “EZ” water because it profoundly excludes solutes. Hence, the buildup of EZ water occurs naturally and spontaneously from environmental energy. Additional energy input creates additional EZ buildup. Of particular significance is the fourth phase’s charge, usually negative (Figure 2). Absorbed radiant energy splits water molecules; the negative moiety forms the building block of the EZ, while the positive moiety binds with ordinary water molecules to form free hydronium ions that diffuse throughout the water. Additional light stimulates more charge separation. This energetic process resembles the first step of photosynthesis. In that initial step, energy from the sun splits water molecules. Chromophores, such as chlorophyll, catalyze water splitting. The process considered here is similar, but more generic: any hydrophilic surface, not just chromophores, can catalyze the splitting, with some surfaces being more effective than others. The assemblage of separated charges resembles a battery. The “water battery” can deliver energy in a similar way to that in which separated charges in plants deliver energy. Plants mostly comprise water, and it is therefore not
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Various talks describe these fresh understandings. One of them is a University of Washington public award lecture http://www.youtube. com/watch?v¼XVBEwn6iWOo. Another was delivered more recently http://www.youtube.com/watch?v¼JnGCMQ8TJ_g. A third is a recent TEDx talk http://youtu.be/i-T7tCMUDXU. A much fuller and deeper presentation appears in the above-mentioned book (Pollack, 2013).
Cell Biol Int 9999 (2014) 1–6 © 2014 International Federation for Cell Biology
Figure 2 Diagrammatic representation of EZ water, negatively charged, and the positively charged bulk water beyond. Hydrophilic surface at left.
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Figure 3 Practically incessant flow occurs through hydrophilic tubes immersed in water.
unexpected that similar energy conversion takes place in water itself. The stored electrical energy in water can drive different kinds of work, including flow. An example is the axial flow through tubes (Rohani and Pollack, 2013; Yu et al., 2013). Merely immersing tubes made of hydrophilic materials into water produces flow through those tubes, similar to the flow of blood through capillaries (Rohani and Pollack, 2013; Yu et al., 2013; Figure 3; and see below). The driving energy comes from radiant energy absorbed by the water, nothing more. Flow may persist undiminished for many hours, even days. The more intense the incident light, the faster the flow (Rohani and Pollack, 2013). This is not a perpetual motion machine because incident radiant energy drives the flow. Even at night, ample infrared energy is available to drive the flow.
Applications in biology This water-based energy conversion framework seems rich with implications. One example is inside the cell, in which more than two-thirds of the volume is water. This translates to >99% of the molecules being water because of their diminutive size compared with the larger molecules in the cell. Modern cell biology considers that 99% of the cell’s molecules are largely background carriers of the “important” molecules of life such as proteins and nucleic acids; in other words, 99% of the cell’s molecules do little more than hold the important molecules like a reservoir. However, EZ water envelops every macromolecule in the cell. Those macromolecules pack so tightly that the enveloping water largely fills the volume of the cell. In other words most cell water is liquid-crystalline, EZ water; it plays a central role in practically everything the cell does (Pollack, 2001). What is new is the role of radiant energy. Incident radiant energy builds EZ water and separates charge, the latter having the potential to power many cellular functions. A possible example is blood flow. Blood easily flows through the larger vessels, but begins to encounter difficulty flowing through the capillaries, which can be narrower than the red blood cells that must pass through them. Red cells need to contort in order to make their way through. Bending those 4
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stiff cells requires lots of driving pressure; yet the pressure gradient across the capillary bed is quite small. The paradox can be resolved if radiant energy helps propel flow through capillaries in the same way that it propels flow through the other hydrophilic tubes. Radiant energy may constitute an unsuspected source of vascular drive, supplementing cardiac pressure. It may also drive flow through lymphatic vessels, and it may propel the streaming that commonly occurs inside cells. None of these physiological flow phenomena have been fully explained. Light-driven flow may therefore provide a key that unlocks multiple doors to a better understanding. Another example of the EZ’s role lies in self-assembly. How do freshly synthesized amino acids self-assemble into proteins? And, how do freshly synthesized proteins selforganize into filaments or vesicles? The EZ shells that envelop those structures bear negative charge. Negatively charged bodies should repel one another, precluding any self-assembly into larger structures; however, molecules do condense into larger structures, and the question is how that can happen. One answer could be the unlike charges that lie in between. Richard Feynman, the legendary Nobel Prize physicist of the late 20th century, understood this principle, opining that: “like-likes-like because of an intermediate of unlikes.” The unlikes are the attractors that make it happen (Figure 4). The like-likes-like principle has been widely appreciated, but also widely ignored: after all, how could like charges conceivably attract? A reason why this simple but powerful concept has been ignored is the difficulty of identifying the source of the unlike charges. We now know that the unlike charges can come from the splitting of water—the negative components building EZ shells, and the corresponding positive components providing the attractors. With enough of these attractors, the negatively charged molecules may
Figure 4 Like-charged entities attract because of an intermediate of opposite charge.
Cell Biol Int 9999 (2014) 1–6 © 2014 International Federation for Cell Biology
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condense into larger structures; i.e., the attractors can promote self-assembly. These two phenomena, functional enhancement by radiant-energy absorption and like-likes-like self-assembly, provide representative examples of the explanatory power of fourth-phase water. They show how water’s fourth-phase can help explain phenomena that otherwise seem inexplicable. Let us now return to the issue of the cell’s electrical potential.
Water and cell charge With some understanding of water’s fourth phase, we return to the question raised earlier: what is responsible for the cell’s negative electrical potential? I have raised questions about the prevailing mechanism, which invokes membrane transporters driving positive charges out of the cell, their re-entry restricted by closed membrane channels. Those mechanisms could potentially suffice, but their acceptance requires meaningful and convincing responses to the fundamental questions raised above. A simple mechanism for the cell’s negative electrical potential follows from the discussion above where I suggested that the responsibility might lie in water. However, H2O has no net charge, so that explanation would seem to be a nonstarter. But EZ water does bear charge. Populating the cell with negatively charged EZ water molecules offers a simple explanation for the cell’s negative electrical potential. A possible counter-argument is that an equal number of positive charges might accompany those negative charges (Figure 2); this would bring electro-neutrality. However, the positive charges act differently; unlike the negatively charge EZ, which clings to macromolecules and remains locked inside the cell, the positive charges are free protons (or hydronium ions). Repelling one another, those positive ions easily exit from the cell. Their exit leaves the remaining cell matrix replete with negative charge (the same for the gel matrix). This explains the cell’s negative charge, as well as the gel’s negative charge. Neither explanation requires any membrane-based mechanisms. The new explanation offers fertile ground for understanding pathologies. Sick cells often have electrical potentials of smaller magnitude than those of normal cells. For example, cancer cells can have negative potentials of 10 mV (Aull, 1967) or 15 mV (Borle and Loveday, 1968) instead of negative 80–100 mV. Anyone routinely measuring electrical potentials knows that when cell negativity falls significantly toward zero, the cell is heading for death. If the EZ supplies the negativity to the cell, then the low negative potential magnitudes in pathological cells reflect insufficient EZ hydration. This implies that proper hydration should drive electrical potential buildup towards normal. Since hydration is necessary for normal protein function (HäusCell Biol Int 9999 (2014) 1–6 © 2014 International Federation for Cell Biology
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singer et al., 1993; Häussinger et al., 1995; Waldegger et al., 1997), this provides a rationale that can explain why hydration (also light) rebuilds function. This explanation also provides a fresh basis for understanding the genesis of action potentials. Textbook explanations rest on the opening and closing of membrane channels. An alternative explanation rests on phase transitions in water, for which abundant evidence has been presented for many cellular functions (Pollack, 2001). Recent studies in muscle tissue amplify this evidence by directly showing a contraction-dependent phase change of water from ordered to partially disordered (Yoo et al., 2014). If ordered water bears negative charge whereas disordered water (H2O) bears no charge, then the change of electrical potential associated with the action potential may be explicable. As the latter thesis needs further study, it gains support from the action of anesthetics. Anesthetics block action potential transmission (supposedly by acting on sodium channels). If action potentials depend on the presence of EZ water, anesthetics should compromise EZ water. This has been confirmed with local anesthetics, including Lidocaine and Bupivocaine (Pollack, 2013), and the general anesthetic, Fluothane (Kundacina and Shi Pollack, unpublished data). Hence, EZ water may be tied intimately with cell action potentials.
The future The cell’s most populous molecule is water. Understanding of water’s properties had been constrained by the common notion that water has only three phases, whereas I have presented evidence for four. Taking into consideration this fourth phase helps account for many of water’s “anomalies,” which now turn into predictable features. Water’s properties become more understandable, as do entities made largely of water, such as the cell. Indeed water’s role in cell biology may be surprisingly central. Until now, these concepts have been applied to biology only in the most rudimentary way, but there is much more to come. Recognizing the abundance of interfacial (EZ) water inside the cell opens the potential for much new understanding. Perhaps pumps and channels may turn out to be more critical than estimated following this better understanding, but at present some pressing questions need to be answered. References Aull F (1967) Measurement of the electrical potential difference across the membrane of the Ehrlich mouse ascites tumor cells. J Cell Physiol 69: 21–32. Borle A, Loveday J (1968) Effects of temperature, potassium and calcium on the HeLa cells. Cancer Res 28: 2401–5.
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Chai B, Yoo H, Pollack GH (2009) Effect of radiant energy on near-surface water. J Phys Chem B 113: 13953–8. Frey-Wyssling A (1953) Submicroscopic morphology of protoplasm. Zeitschrift Für Pflanzenernährung, Düngung Bodenkunde 68: 75–6. Häussinger D, Lang FW, Gerok W (1995) Regulation of metabolism by changes in cellular hydration. Am J Clin Nutr 14: 4–12. Häussinger D, Roth E, Lang F, Gerok W (1993) Celluar hydration state: an important determinant of protein catabolism in health and disease. Lancet 341: 1330–2. Laub KR, Witschas K, Blicher A, Madsen SB, Lückhoff A, Heimburg T (2012) Comparing iron conductance recordings of synthetic lipid bilayers with cell membranes containing TRP channels. Biochimica Biophysica Acta 1818: 1123–34. Lev A, Korchev Y, Rostovtseva T, Bashford C, Edmonds D, Pasternak C (1993) Rapid switching of ion current in narrow pores: implications for biological ion channels. Proc Biol Sci 252: 187–92. Ling GN (1997) Debunking the alleged resurrection of the sodium pump hypothesis. Physiol Chem Phys Med NMR 29: 123–98. Neher E, Sackmann B, Steinbach JH (1978) The extracellular patch clamp: a method for resolving currents through individual open channels in biological membranes. Pflügers Arch 375: 219–28. Pollack GH (2013) The fourth phase of water: beyond solid, liquid, and vapor. Seattle, WA: Ebner & Sons.
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Pollack GH (2001) Cells, gels and the engines of life. Seattle, WA: Ebner & Sons. Rohani M, Pollack GH (2013) Flow through horizontal tubes submerged in water in the absence of a pressure gradient: mechanistic considerations. Langmuir 29(22): 6556–61. Sachs F, Qin F (1993) Gated, ion-selective channels observed with patch pipettes in the absence of membranes: novel properties of gigaseal. Biophys J 65: 1101–7. Waldegger S, Busch G, Kaba N, Zempel G, Ling H, Heidland A, Häussinger D, Lang F (1997) Effect of cellular hydration on protein metabolism. Miner Electrolyte Metab 23: 201–5. Woodbury D (1989) Pure Lipid vesicles can induce channel-like conductances in planar bilayers. J Membrane Biol 109: 145–50. Yoo H, Nagronyak E, Das R, Wexler A, Pollack GH (2014) Contraction-induced changes in hydrogen bonding of muscle hydration water. J Phys Chem 5: 947–52. Yu A, Carlson P, Pollack GH (2013) Unexpected axial flow through hydrophilic tubes: implication for energetics of water. Eur Phys J Special Topics 223: 947–58. Zheng J, Chin W, Khijniak E, Khijniak E, Jr., Pollack GH (2006) Surfaces and interfacial water: evidence that hydrophilic surfaces have long-range impact. Adv Colloid Interface Sci 127: 19–27. Zhou Y, Morais-Cabral J, Kaufman A, MacKinnon R (2001) Chemistry of ion coordination and hydration revealed by a Kþ channel-Fab complex at 2.0 A resolution. Nature 414: 43–8. Received 16 September 2014; accepted 17 September 2014.
Cell Biol Int 9999 (2014) 1–6 © 2014 International Federation for Cell Biology
COMMENTARY
Cell electrical properties: reconsidering the origin of the electrical potential Gerald H. Pollack* Department of Bioengineering, University of Washington, Seattle 98195, WA, USA
Introduction The interior of the cell commonly has a negative electrical potential of 50–100 mV, a fact known for more than a half century. To create this potential difference, the ionic contents inside the cell must differ from those outside the cell. I present evidence that the potential difference arises, at least in part, from the unique characteristics of intracellular water, rather than from the action of membrane transporters (pumps) and channels.
Pump and channel issues Textbooks continue to espouse the long-held view that certain ions (and other substances) get pumped into or out of the cell, and subsequently re-enter and exit through selective channels. These membrane-based processes are widely presumed to create intracellular negativity. Pump and channel mechanisms have become so deeply ingrained into modern cell biology as to be essentially axiomatic. A number of criticisms lodged against those mechanisms have seldom been seriously addressed. One of them has focused on membrane pumps. Pumps require energy, and Ling (1997) showed that the cell could not possibly generate the energy required to power even the sodium pump, let alone the many other membrane pumps that have since been claimed to exist. Ling reviews the evidence on his website www.GilbertLing.org and argues that there has been no meaningful response to this proposal. Energy issues notwithstanding, the broad scientific community, in embracing the pump concepts, are confident of its explanatory power and consequent adequacy. However, the above-mentioned challenge remains unaddressed, and until it is, some doubt must linger. Membrane channels occupy a similar place in contemporary thinking, with a huge body of evidence seeming to underlie the existence of selective channels, their number now exceeding 400, with each channel being selective for a
different ion or solute. Therapeutic pharmaceutical agents commonly are claimed to act on one or other of these channels, thereby producing a physiological response. Like pump mechanisms, channels have become broadly accepted. Two challenges have yet to receive meaningful responses (Pollack, 2001). The first questions the original evidence for the existence of single ion-selective channels, which came from the classic paper by Neher et al. (1978) who used a patch of membrane. When the potential difference across that patch was raised beyond a certain threshold, pulse-like currents of consistent magnitude began flowing through the patch. Channel openings and closings were assumed to account for these discrete currents. However, similar pulselike currents appear in patches that have no channels, as has been corroborated by Sachs and Qin (1993), Lev et al. (1993) and Woodbury (1989). The original papers of Neher and Sackmann hinted at such difficulties, which were quickly dismissed. In an extensive series of experiments, Thomas Heimburg’s group at the Nils Bohr Institute more recently reported similar results (Laub et al., 2012); i.e., membranes with TRP (transient receptor potential) channels show conductance behavior indistinguishable from those of synthetic lipid bilayers, containing no channels at all. (A representative result is given in Figure 1). One would think this evidence to be troubling. As the presence of pulse-like currents of consistent amplitude constitutes the primary evidence for the existence of selective membrane channels, evidence of similar behavior in specimens with no channels suggests prudence. The second channel-related issue is conceptual. Suppose the membrane contains 400 channels, each selective for a particular solute or ion. When a particular channel opens, its respective solute passes through while other solutes remain excluded, and the channel then closes. This seems simple and elegant, but there is a problem. Solutes are of varied size and nature. Consider the channel that is selective for the largest of the 400 solutes. When that channel opens, what prevents the smaller solutes from passing through at the
Corresponding author: e-mail: [email protected]
Cell Biol Int 9999 (2014) 1–6 © 2014 International Federation for Cell Biology
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Figure 1 Patch clamp recording of a synthetic lipid membrane with no proteins or channels. Eight representative 2.5 s segments out of a 30-min recording. From Laub et al., 2002.
same time? If the channel is genuinely selective, it has to prevent all 399 smaller solutes from passing through. The 399th largest must prevent all 398 smaller ones from passing through, etc. It can be likened to the dog door, which can flap open to allow your pet dog to pass through, but it would not prevent a cat, ferret, and mouse from passing through at the same time. This theoretical conundrum has received at least some attention. In the classical work of MacKinnon and his colleagues (Zhou et al., 2001), the authors explained how a channel might distinguish between sodium and potassium ions, a finding important enough to have earned the senior author a Nobel Prize. It went partway towards dealing with the selectivity challenge; however, the treatment dealt principally with two ions of similar character. The selectivity problem is more extensive, for how could a channel large enough to pass a bulky molecule exclude a molecule one hundredth of its cross-section, such as water? If we take the fallback position and admit that the large channel does not really exclude all those smaller molecules, and that they may actually pass through, down their respective concentration gradients, why does the cell need channels devoted exclusively to those smaller molecules? If large channels can pass water, why does the cell need an aquaporin channel devoted exclusively to water, which seems like redundancy? The challenge of selectivity has to be addressed; at the very least, a convincing theory has to explain how a channel can permit passage of a particular 2
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solute, while excluding all solutes of smaller diameter, including some that are much smaller; otherwise the selective channel concept seems bereft of theoretical underpinning. In sum, it is thought that the cell’s negative electrical potential arises from the action of membrane pumps and channels. For most investigators these mechanisms seem acceptable and adequate, although still based on some assumptions and possibly shaky premises. However, the conceptual and experimental challenges raised above to the fundamental underpinning of these mechanisms needs to be fully addressed. For those who consider these issues minor compared to all the positive evidence, consider the conundrum of crowding. Membrane channels and pumps are continually being identified. The channel count may exceed 400, but the transporter count totals 1,000 (some colleagues put the “unofficial” transporter count at several thousand). To operate effectively, each of these entities must appear multiple times on the membrane of each cell. Even if some functions are shared, nevertheless, tens of thousands of pumps/channels must crowd into the cell membrane, along with the various receptors and other functional entities—it must be hugely crowded. The crowding problem grows more acute with time; if, as seems to be the case, the membrane’s surface area remains relatively fixed while additional channels/pumps appear, for example, for each newly synthesized drug, will membrane space eventually run out, unless existing channels are commandeered? These are not complicated arguments, but do they imply a logical conundrum. How certain we can be that the prevailing paradigm explaining the cell’s handling of ions is adequate? Can we be certain that the electrical potential of the cell is generated by the actions of pumps and channels?
Gel electrical potentials and cell electrical potentials The first inkling that the cell’s negative electrical potential might involve water came from a simple observation: the similarity of cells and gels. The cell cytoplasm is gel-like. Many cell biologists have routinely worked with “demembranated” cells. Following demembranation by mechanical or chemical means, the rest of the cell hangs together like Jell-O; it does not dissolve into the surrounding milieu as would an aqueous solution. The gel-like nature of the cytoplasm has long been established (Frey-Wyssling, 1953), and further evidence gives additional force to that consistency (Pollack, 2001). Electrical features of cells and gels are also similar; one can measure the gel’s electrical potential (more accurately the potential difference between inside and outside of the gel) by sticking a microelectrode in a gel. It is remarkable that the electrical potential of a gel can be similar to the electrical Cell Biol Int 9999 (2014) 1–6 © 2014 International Federation for Cell Biology
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potential of the cell: both can have negative potentials of the order of roughly 100 mV (Pollack, 2001; Zheng et al., 2006). Yet, the gel has no membrane, nor any channels or pumps. Either different mechanisms govern the gel’s and the cell’s potential or surface membranes have little or nothing to do with the cell’s electrical potential. We previously thought that both of these negative potentials might arise from the internal negatively charged proteins/polymers. The negativity of these solids might explain the observed negativity. This supposition must, at least partially, be valid. However, some difficulty arose when trying to relate this negativity to the rapid shifts of electrical potential occurring in excitable cells, as manifested in action potentials. The protein/polymer charge explanation seems inadequate; the problem is therefore more complicated than this alternative simple explanation indicates. Thus, we have to consider more carefully the role of water, by far the most populous molecule inside the cell. The idea of water’s contribution became compelling when it was discovered that water could bear negative charge. I will return to the relation between water’s negative charge and the cell’s negative potential after briefly explaining the unexpected finding of water’s negativity and some of its consequences.
A “New” phase of water? There is a fourth phase of water (Pollack, 2013; The Fourth Phase of Water: Beyond Solid, Liquid and Vapor; see also footnote), which is found next to water-loving (hydrophilic) surfaces such as proteins and other charged macromolecules. The phase is more extensive than originally conjectured, projecting out from surfaces by many molecular layers. It exists almost everywhere throughout nature, including the cell. Details of this newly identified phase of water have been described in many papers, and many basic findings have been compiled in the above-mentioned book, most of which require exploration in their nature and applications for technology. The book also deals with water’s many known anomalies, turning them into explained features. The existence of a fourth phase may seem unexpected; however, that should not be entirely so. A century ago, the respected physical chemist Sir William Hardy argued for the existence of a fourth phase; and a number of authors over the years have found evidence for some kind of “ordered” or “structured” phase of water. New
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experimental evidence not only confirms the existence of such an ordered, liquid-crystalline phase, but also details its remarkable properties. The energy for building this water structure comes ultimately from the sun. Radiant energy converts ordinary bulk water into ordered water, building this ordered zone inside and outside of biology. All wavelengths, ranging from UV through visible to infrared, suffice. Near-infrared and mid-infrared energy are the most capable (Chai et al., 2009). Water absorbs infrared energy freely from the environment and uses it to convert bulk water into liquid crystalline water covering hydrophilic surfaces of all types—which we call “exclusion zone” or “EZ” water because it profoundly excludes solutes. Hence, the buildup of EZ water occurs naturally and spontaneously from environmental energy. Additional energy input creates additional EZ buildup. Of particular significance is the fourth phase’s charge, usually negative (Figure 2). Absorbed radiant energy splits water molecules; the negative moiety forms the building block of the EZ, while the positive moiety binds with ordinary water molecules to form free hydronium ions that diffuse throughout the water. Additional light stimulates more charge separation. This energetic process resembles the first step of photosynthesis. In that initial step, energy from the sun splits water molecules. Chromophores, such as chlorophyll, catalyze water splitting. The process considered here is similar, but more generic: any hydrophilic surface, not just chromophores, can catalyze the splitting, with some surfaces being more effective than others. The assemblage of separated charges resembles a battery. The “water battery” can deliver energy in a similar way to that in which separated charges in plants deliver energy. Plants mostly comprise water, and it is therefore not
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Various talks describe these fresh understandings. One of them is a University of Washington public award lecture http://www.youtube. com/watch?v¼XVBEwn6iWOo. Another was delivered more recently http://www.youtube.com/watch?v¼JnGCMQ8TJ_g. A third is a recent TEDx talk http://youtu.be/i-T7tCMUDXU. A much fuller and deeper presentation appears in the above-mentioned book (Pollack, 2013).
Cell Biol Int 9999 (2014) 1–6 © 2014 International Federation for Cell Biology
Figure 2 Diagrammatic representation of EZ water, negatively charged, and the positively charged bulk water beyond. Hydrophilic surface at left.
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Figure 3 Practically incessant flow occurs through hydrophilic tubes immersed in water.
unexpected that similar energy conversion takes place in water itself. The stored electrical energy in water can drive different kinds of work, including flow. An example is the axial flow through tubes (Rohani and Pollack, 2013; Yu et al., 2013). Merely immersing tubes made of hydrophilic materials into water produces flow through those tubes, similar to the flow of blood through capillaries (Rohani and Pollack, 2013; Yu et al., 2013; Figure 3; and see below). The driving energy comes from radiant energy absorbed by the water, nothing more. Flow may persist undiminished for many hours, even days. The more intense the incident light, the faster the flow (Rohani and Pollack, 2013). This is not a perpetual motion machine because incident radiant energy drives the flow. Even at night, ample infrared energy is available to drive the flow.
Applications in biology This water-based energy conversion framework seems rich with implications. One example is inside the cell, in which more than two-thirds of the volume is water. This translates to >99% of the molecules being water because of their diminutive size compared with the larger molecules in the cell. Modern cell biology considers that 99% of the cell’s molecules are largely background carriers of the “important” molecules of life such as proteins and nucleic acids; in other words, 99% of the cell’s molecules do little more than hold the important molecules like a reservoir. However, EZ water envelops every macromolecule in the cell. Those macromolecules pack so tightly that the enveloping water largely fills the volume of the cell. In other words most cell water is liquid-crystalline, EZ water; it plays a central role in practically everything the cell does (Pollack, 2001). What is new is the role of radiant energy. Incident radiant energy builds EZ water and separates charge, the latter having the potential to power many cellular functions. A possible example is blood flow. Blood easily flows through the larger vessels, but begins to encounter difficulty flowing through the capillaries, which can be narrower than the red blood cells that must pass through them. Red cells need to contort in order to make their way through. Bending those 4
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stiff cells requires lots of driving pressure; yet the pressure gradient across the capillary bed is quite small. The paradox can be resolved if radiant energy helps propel flow through capillaries in the same way that it propels flow through the other hydrophilic tubes. Radiant energy may constitute an unsuspected source of vascular drive, supplementing cardiac pressure. It may also drive flow through lymphatic vessels, and it may propel the streaming that commonly occurs inside cells. None of these physiological flow phenomena have been fully explained. Light-driven flow may therefore provide a key that unlocks multiple doors to a better understanding. Another example of the EZ’s role lies in self-assembly. How do freshly synthesized amino acids self-assemble into proteins? And, how do freshly synthesized proteins selforganize into filaments or vesicles? The EZ shells that envelop those structures bear negative charge. Negatively charged bodies should repel one another, precluding any self-assembly into larger structures; however, molecules do condense into larger structures, and the question is how that can happen. One answer could be the unlike charges that lie in between. Richard Feynman, the legendary Nobel Prize physicist of the late 20th century, understood this principle, opining that: “like-likes-like because of an intermediate of unlikes.” The unlikes are the attractors that make it happen (Figure 4). The like-likes-like principle has been widely appreciated, but also widely ignored: after all, how could like charges conceivably attract? A reason why this simple but powerful concept has been ignored is the difficulty of identifying the source of the unlike charges. We now know that the unlike charges can come from the splitting of water—the negative components building EZ shells, and the corresponding positive components providing the attractors. With enough of these attractors, the negatively charged molecules may
Figure 4 Like-charged entities attract because of an intermediate of opposite charge.
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condense into larger structures; i.e., the attractors can promote self-assembly. These two phenomena, functional enhancement by radiant-energy absorption and like-likes-like self-assembly, provide representative examples of the explanatory power of fourth-phase water. They show how water’s fourth-phase can help explain phenomena that otherwise seem inexplicable. Let us now return to the issue of the cell’s electrical potential.
Water and cell charge With some understanding of water’s fourth phase, we return to the question raised earlier: what is responsible for the cell’s negative electrical potential? I have raised questions about the prevailing mechanism, which invokes membrane transporters driving positive charges out of the cell, their re-entry restricted by closed membrane channels. Those mechanisms could potentially suffice, but their acceptance requires meaningful and convincing responses to the fundamental questions raised above. A simple mechanism for the cell’s negative electrical potential follows from the discussion above where I suggested that the responsibility might lie in water. However, H2O has no net charge, so that explanation would seem to be a nonstarter. But EZ water does bear charge. Populating the cell with negatively charged EZ water molecules offers a simple explanation for the cell’s negative electrical potential. A possible counter-argument is that an equal number of positive charges might accompany those negative charges (Figure 2); this would bring electro-neutrality. However, the positive charges act differently; unlike the negatively charge EZ, which clings to macromolecules and remains locked inside the cell, the positive charges are free protons (or hydronium ions). Repelling one another, those positive ions easily exit from the cell. Their exit leaves the remaining cell matrix replete with negative charge (the same for the gel matrix). This explains the cell’s negative charge, as well as the gel’s negative charge. Neither explanation requires any membrane-based mechanisms. The new explanation offers fertile ground for understanding pathologies. Sick cells often have electrical potentials of smaller magnitude than those of normal cells. For example, cancer cells can have negative potentials of 10 mV (Aull, 1967) or 15 mV (Borle and Loveday, 1968) instead of negative 80–100 mV. Anyone routinely measuring electrical potentials knows that when cell negativity falls significantly toward zero, the cell is heading for death. If the EZ supplies the negativity to the cell, then the low negative potential magnitudes in pathological cells reflect insufficient EZ hydration. This implies that proper hydration should drive electrical potential buildup towards normal. Since hydration is necessary for normal protein function (HäusCell Biol Int 9999 (2014) 1–6 © 2014 International Federation for Cell Biology
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singer et al., 1993; Häussinger et al., 1995; Waldegger et al., 1997), this provides a rationale that can explain why hydration (also light) rebuilds function. This explanation also provides a fresh basis for understanding the genesis of action potentials. Textbook explanations rest on the opening and closing of membrane channels. An alternative explanation rests on phase transitions in water, for which abundant evidence has been presented for many cellular functions (Pollack, 2001). Recent studies in muscle tissue amplify this evidence by directly showing a contraction-dependent phase change of water from ordered to partially disordered (Yoo et al., 2014). If ordered water bears negative charge whereas disordered water (H2O) bears no charge, then the change of electrical potential associated with the action potential may be explicable. As the latter thesis needs further study, it gains support from the action of anesthetics. Anesthetics block action potential transmission (supposedly by acting on sodium channels). If action potentials depend on the presence of EZ water, anesthetics should compromise EZ water. This has been confirmed with local anesthetics, including Lidocaine and Bupivocaine (Pollack, 2013), and the general anesthetic, Fluothane (Kundacina and Shi Pollack, unpublished data). Hence, EZ water may be tied intimately with cell action potentials.
The future The cell’s most populous molecule is water. Understanding of water’s properties had been constrained by the common notion that water has only three phases, whereas I have presented evidence for four. Taking into consideration this fourth phase helps account for many of water’s “anomalies,” which now turn into predictable features. Water’s properties become more understandable, as do entities made largely of water, such as the cell. Indeed water’s role in cell biology may be surprisingly central. Until now, these concepts have been applied to biology only in the most rudimentary way, but there is much more to come. Recognizing the abundance of interfacial (EZ) water inside the cell opens the potential for much new understanding. Perhaps pumps and channels may turn out to be more critical than estimated following this better understanding, but at present some pressing questions need to be answered. References Aull F (1967) Measurement of the electrical potential difference across the membrane of the Ehrlich mouse ascites tumor cells. J Cell Physiol 69: 21–32. Borle A, Loveday J (1968) Effects of temperature, potassium and calcium on the HeLa cells. Cancer Res 28: 2401–5.
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Chai B, Yoo H, Pollack GH (2009) Effect of radiant energy on near-surface water. J Phys Chem B 113: 13953–8. Frey-Wyssling A (1953) Submicroscopic morphology of protoplasm. Zeitschrift Für Pflanzenernährung, Düngung Bodenkunde 68: 75–6. Häussinger D, Lang FW, Gerok W (1995) Regulation of metabolism by changes in cellular hydration. Am J Clin Nutr 14: 4–12. Häussinger D, Roth E, Lang F, Gerok W (1993) Celluar hydration state: an important determinant of protein catabolism in health and disease. Lancet 341: 1330–2. Laub KR, Witschas K, Blicher A, Madsen SB, Lückhoff A, Heimburg T (2012) Comparing iron conductance recordings of synthetic lipid bilayers with cell membranes containing TRP channels. Biochimica Biophysica Acta 1818: 1123–34. Lev A, Korchev Y, Rostovtseva T, Bashford C, Edmonds D, Pasternak C (1993) Rapid switching of ion current in narrow pores: implications for biological ion channels. Proc Biol Sci 252: 187–92. Ling GN (1997) Debunking the alleged resurrection of the sodium pump hypothesis. Physiol Chem Phys Med NMR 29: 123–98. Neher E, Sackmann B, Steinbach JH (1978) The extracellular patch clamp: a method for resolving currents through individual open channels in biological membranes. Pflügers Arch 375: 219–28. Pollack GH (2013) The fourth phase of water: beyond solid, liquid, and vapor. Seattle, WA: Ebner & Sons.
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Pollack GH (2001) Cells, gels and the engines of life. Seattle, WA: Ebner & Sons. Rohani M, Pollack GH (2013) Flow through horizontal tubes submerged in water in the absence of a pressure gradient: mechanistic considerations. Langmuir 29(22): 6556–61. Sachs F, Qin F (1993) Gated, ion-selective channels observed with patch pipettes in the absence of membranes: novel properties of gigaseal. Biophys J 65: 1101–7. Waldegger S, Busch G, Kaba N, Zempel G, Ling H, Heidland A, Häussinger D, Lang F (1997) Effect of cellular hydration on protein metabolism. Miner Electrolyte Metab 23: 201–5. Woodbury D (1989) Pure Lipid vesicles can induce channel-like conductances in planar bilayers. J Membrane Biol 109: 145–50. Yoo H, Nagronyak E, Das R, Wexler A, Pollack GH (2014) Contraction-induced changes in hydrogen bonding of muscle hydration water. J Phys Chem 5: 947–52. Yu A, Carlson P, Pollack GH (2013) Unexpected axial flow through hydrophilic tubes: implication for energetics of water. Eur Phys J Special Topics 223: 947–58. Zheng J, Chin W, Khijniak E, Khijniak E, Jr., Pollack GH (2006) Surfaces and interfacial water: evidence that hydrophilic surfaces have long-range impact. Adv Colloid Interface Sci 127: 19–27. Zhou Y, Morais-Cabral J, Kaufman A, MacKinnon R (2001) Chemistry of ion coordination and hydration revealed by a Kþ channel-Fab complex at 2.0 A resolution. Nature 414: 43–8. Received 16 September 2014; accepted 17 September 2014.
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