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viii

C CONTENTS

ACKNOWLEDGEMENTS vi

PREFACE x

SECTION I. TOWARD GROUND TRUTH 1This section deals with generally accepted views of cell biology. By peel-ing back layers of assumption, it attempts to get at the core of truth. Inthe end, an unorthodox conclusion is drawn about the nature of the cell.

1. Debunking Myths 32. The Croak of the Dying Cell 253. Cytoplasmic Discomfort 39

SECTION II. BUILDING FROM BASICS 51This section pursues elementary cell function, with emphasis on waterand protein surfaces. We see how the interaction of these two elementsgives rise to ion partitioning, cell potential, and several other of the mostbasic features of cell physiology in ways that differ from current views.

4. Water 535. Solutes 776. Ions 877. Cell Potentials 99

ix

SECTION III. AN HYPOTHESIS FOR CELL FUNCTION 111Building on previous chapters, this section advances the hypothesis thatcell function resembles gel function. It examines the role of the phase-transition in gels, and considers the potential for a similar role in cells.

8. Phase Transition: A Mechanism for Action 113

SECTION IV. APPROACHING CELL DYNAMICS 131This section pursues details of active cell function. Building onprevious chapters, it explores the possibility that diverse cellular actionsare mediated by a common mechanism—the phase-transition.

9. Secretion 13310. The Action Potential 14511. Transport 16312. Transport with Flair 18513. Cell Division 20714. Muscle Contraction

SECTION V. TYING LOOSE ENDS 249This section considers issues common to the preceding chapters. It be-gins with cellular evolution and energetics, and then moves on to ex-plore the underlying themes that integrate the material of the book.

15. Energy 25116. A New Paradigm for Cell Function 267

REFERENCES 285

INDEX 299

225

x

PREFACE

This book is about the cell and how it functions. Books about cell func-tion have become commonplace, but the approach taken here is not atall common. It builds on fundamental principles of physics and chemis-try rather than on accepted higher-level constructs. The premise is thatnature’s functional paradigms are elegant, simple, and probably far lessconvoluted than current wisdom purports them to be. The book’s goal isto identify these simple paradigms from physical chemical basics.

The story begins with a curious scene described in the standard cell-biology textbook by Alberts et al. (first edition, p. 604). A crawling cellsomehow manages to get stuck onto the underlying surface. The leadingedge continues to press on, tears free, and continues its journey onwardas though oblivious to the loss of its rear end.

Something about this scenario does not seem right. We have been taughtthat cell function depends on the integrity of the cell membrane; yet themembrane of this cell has been ripped asunder and the cell continues tofunction as though none of this matters. It’s business as usual—eventhough the cell has effectively been decapitated.

When first exposed to this scenario, I reacted the way you may be react-ing—the cell must somehow reseal. The membrane must spread over thejagged surfaces, covering the wound and restoring the cell’s integrity. Iwas inclined to accept this seemingly flimsy account even though I couldnot understand how a bilayer membrane built of molecules with fixedlateral packing could spread over half again as much area. Nevertheless,it seemed expedient to shelve this anomaly and press on with more im-mediate matters; someone else could figure out what might be going on.

P

xi

With time, it became clear that the survival of the decapitated cell wasbut one example of a set of related anomalies. Cells survive sundry in-sults equivalent to being guillotined, drawn and quartered, and shot fullof holes with electrical bullets (Chapter 2). If the ruptured membranedoes not reseal—and no evidence I’ve seen convinces me that “resealing”is any more than a conveniently invoked expedient—the implication isthat membrane integrity may be less consequential than presumed.

From this realization springs the early material of this book. If mem-brane integrity is not essential, then what holds the contents inside thecell? If you are willing to consider the cytoplasm as a gel instead of anordinary aqueous solution, then an answer may be at hand, for gels don’tnecessarily disintegrate when sliced; the contents will not spill out.

The gel-like nature of the cytoplasm forms the foundation of this book.Biologists acknowledge the cytoplasm’s gel-like character, but textbooksnevertheless build on aqueous solution behavior. A gel is quite differentfrom an aqueous solution—it is a matrix of polymers to which water andions cling. That’s why gelatin desserts retain water, and why a crackedegg feels gooey.

The concept of a gel-like cytoplasm turns out to be replete with power.It accounts for the characteristic partitioning of ions between the insideand outside of the cell (Chapter 6). It also explains the cell’s electricalpotential: potentials of substantial magnitude can be measured in gels aswell as in demembranated cells (Chapter 7). Thus, the gel-like characterof the cytoplasm accounts for the basic features of cell biophysics.

The focus then shifts from statics to dynamics. Here again the questionis whether adequate explanations can emerge from the cell’s gel-like char-acter. Contrary to the common perception, gels are not inert. Withmodest prompting, polymer gels undergo structural transitions that canbe as profound as the change from ice to water, which is why they areclassified as phase-transitions. Polymer-gel phase-transitions are com-monly exploited in everyday products ranging from time-release pills todisposable diapers. They have immense functional potential.

xii

Whether the cell exploits such functional potential is the focus of thebook’s second half. I explore the possibility that the phase-transitioncould be a common denominator of cell function. By cell function Imean material transport, motility, division, secretion, communication,contraction—among other essential functions.

So there we stand. Driven by the notion of simplicity, I suggest thatmuch of cell biological function may be governed by a single unifyingmechanism—the phase-transition. This is a heady notion, which shouldbe met with due skepticism. Nevertheless, it is a course that followsnaturally if the cytoplasm and its working organelles are gel-like, for thephase-transition is the gel’s central functional agent. Whether ordinarygels and cytoplasmic gels operate by the same working principle is thetheme of this book.

***

The book is designed for those with minimal background in biology. Itcould have been written for experts, rich with theory and dense withdetail. My experience, however, is that too-deep immersion often ob-scures the underlying logic—or lack of it. The strategy is to expose thematerial to scrutiny by focusing on the underlying principles. As such,the material should pose little difficulty for students in any of the physi-cal, biological, or engineering sciences—graduate or undergraduate. Infact, the response to drafts convinces me that the material should be ac-cessible to any intelligent person with open-minded curiosity.

To keep the text streamlined, I have taken a middle ground between theextensive referencing characteristic of scholarly works and the absence ofany referencing characteristic of lay books. References seemed necessaryin controversial areas, but I felt free to omit them in areas I felt weremore standard. I hope this omission will be forgiven. I also hope to beforgiven for having glossed over work that may have consumed someone’slifetime, or for having failed to cite some work that may seem conflict-ing. I can assure you that any such omissions were not purposeful.

I may have conveyed the impression that everything contained in thisbook is original, but that is not the case. The physical features of the

xiii

protein-water-ion complex have been elaborated early on by a cadre ofscientific pioneers including Albert Szent-Györgyi, A. S. Troshin, andGilbert Ling. Ling in particular has spent his long career courageouslyadvancing scientific frontiers in the face of frequent derision for the ap-parent extremity of his views. In my estimate the evidence powerfullysupports his constructs. Indeed, the threads of his ideas weave the veryfabric of this book, particularly the early chapters. The material on phase-transitions rests on the seminal contributions of the late Toyo Tanaka ofMIT, who elaborated the phase-transitions’ underlying mechanisms andforecast their biological relevance. Largely, the book ties together natu-ral principles advanced by others, adding a few thoughts here and thereto integrate these principles and broaden their relevance.

The end result is a fresh view of how cells function. Fresh views do notsit well with scientific establishments and I am not naïve enough to thinkthat the ideas offered here will be embraced with enthusiasm, even ifsome features may be deemed attractive. In the interest of progress, how-ever, maintaining a competing paradigm alongside the orthodox one canbe advantageous, as shortcomings in either can be more readily exposed.But the challenge of dual maintenance is not easy—no easier perhapsthan the challenge of having two lovers and remaining faithful to both atthe same time. It is nevertheless in this constructive spirit that this freshparadigm is offered.

GHPSeattle, November, 2000

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1

SECTION ITOWARD GROUND TRUTH

This section deals with generally accepted views of cell biology. By peeling back layers of assumption, it at-tempts to get at the core of truth. In the end, an unorthodox conclusion is drawn about the nature of the cell.

2

Epicycles

Debunking Myths 3

1CHAPTER 1

DEBUNKING MYTHS

Long ago, scientists believed that the center of the universe was the earth:The sun could be seen to traverse the heavens, so it was logical to con-clude that the earth must lie at the center point.

But this view eventually encountered difficulties. As the growth of math-ematics increased the power of astronomy, it became possible to com-pute orbital pathways. The planets’ paths around the earth turned outto be less simple than anticipated; each planet followed an orbit calledan epicycle (Figure, opposite), which was sufficiently intricate to implythat something was surely amiss.

What was amiss is no longer a mystery. Although the persistent notionof an earth-centered universe may gratify our collective egos, Galileoshowed that it was the sun that held this honor. With the sun at thesolar system’s center, orbital paths no longer required complex epicycles;they became a lot simpler. What had earlier seemed a reasonable hy-pothesis supported by seemingly indisputable visual observation, turnedout to be dead wrong. A complicated paradigm was replaced by a sim-pler one.

IS LIFE REALLY ANY DIFFERENT NOW?

In the field of cell biology at least, complicated paradigms raise similarconcern. On the surface everything seems to be in order. Virtually allknown cellular processes are by now accounted for by well-describedmechanisms: ions flow through channels; solutes are transported by

4

pumps; vesicles are moved by motors; etc. For every problem there is asolution. But as we shall see as we probe beneath the surface of thesesolutions, a bewildering level of complexity hints at a situation that couldparallel the epicycles.

I propose to step back and regroup. For genuine progress, foundationalconcepts must be unquestionably sound; otherwise an edifice of under-standing may rise over a crevasse of uncertainty—no apparent problemuntil the edifice grows weighty enough to crack the foundation and tumbleinto the abyss. Firm ground needs to be identified.

I begin by considering two elements thought to be fundamental to cellfunction: membrane pumps and channels. Pumps transport solutes acrossthe cell boundary against their respective concentration gradients. Chan-nels permit the solutes to trickle back in the opposite direction. Througha balance between pump-based transport and channel-based leakage, thecharacteristic partitioning of solutes and ions is thought to be established.

Table 1.1. Concentration of principal ions inside and outside of a typical

mammalian cell

ExtracellularConcentration

(mM)

145

5

110

Ion MolecularWeight

IntracellularConcentration

(mM)

Na

K

Cl

23

39

35.5

5-15

140

5-15

Debunking Myths 5

Thus, potassium concentration is relatively higher inside the cell, andsodium is relatively higher outside (Table 1.1).

That pumps and channels exist seems beyond doubt—or to put it moreprecisely, the existence of proteins with pump-like or channel-like fea-tures cannot be doubted. Genes coding for these proteins have beencloned, and the proteins themselves have been exhaustively studied. Therecan be no reason why their existence might be challenged.

Where some question could remain is in the functional role of these pro-teins. What I will be considering in this chapter is whether these pro-teins really mediate ion partitioning. Because a “pump” protein insertedinto an artificial membrane can translocate an ion from one side of themembrane to the other, can we be certain that ion partitioning in theliving cell necessarily occurs by pumping?

This task of checking this presumption may in this case be approachedthrough the portal of historical perspective. Scientists on the frontieroften dismiss history as irrelevant but in this particular instance a brieflook into the trail of discovery is especially revealing.

ORIGINS

The emergence of pumps and channels was preceded by the concept ofthe cell membrane. The latter arose during the era of light microscopy,prior to the time any such membrane could actually be visualized. Bi-ologists of the early nineteenth century observed that a lump of cyto-plasm, described as a “pulpy, homogeneous, gelatinous substance”(Dujardin, 1835) did not mix with the surrounding solution.

To explain why this gelatinous substance did not dissolve, the idea arosethat it must be enveloped by a water-impermeant film. This film couldprevent the surrounding solution from permeating into the cytoplasmand dissolving it. The nature of the membranous film had two suggested

6

variants. Kühne (1864) envisioned it as a layer of coagulated protein,while Schültze (1863) imagined it as a layer of condensed cytoplasm.Given the experimental limitations of the era, the nature of the putativefilm, still not visualizable, remained uncertain.

The idea of an invisible film was nevertheless attractive to many of theera’s scientists, and was increasingly conferred with special attributes.Thus, Theodore Schwann (1839) viewed this film as “prior in impor-tance to its contents.” The membrane grew in significance to becomethe presumed seat of much of the cell’s activity. Yet this view was notaccepted by all. Max Schültze, often referred to as the father of modernbiology, discounted the evidence for a cytoplasmic film altogether, andinstead regarded cells as “membraneless little lumps of protoplasm witha nucleus” (Schültze, 1861). In spite of Schültze’s prominence, the con-cept of an enveloping membrane held firm.

The modern idea that the membrane barrier might be semi-permeablecame from the plant physiologist Wilhelm Pfeffer. Pfeffer was aware ofthe ongoing work of Thomas Graham (1861) who had been studyingcolloids, which are large molecules suspended indefinitely in a liquidmedium—e.g., milk. According to Graham’s observations, colloids couldnot pass through dialysis membranes although water could. To Pfeffer,colloids seemed to resemble the cytoplasm. If the dialysis membranewere like the cell membrane, Pfeffer reasoned, the cell interior wouldnot dissipate into the surrounding fluid even though the membrane mightstill be water-permeable. Thus arose the idea of the semi-permeable mem-brane.

Pfeffer took up the semi-permeable membrane idea and pursued it. Hecarried out experiments on membrane models made of copper ferrocya-nide, which acted much like dialysis membranes in that they could passwater easily but solutes with great difficulty. It was on these experimentsthat Pfeffer based the modern cell-membrane theory (Pfeffer, 1877). Themembrane at this stage was presumed permeable to water, but little else.

Debunking Myths 7

Although Pfeffer’s theory held for some time, it suffered serious setbackswhen substances presumed unable to cross the membrane turned out tocross. The first and perhaps most significant of these substances waspotassium. The recognition, in the early twentieth century, that potas-sium could flow into and out of the cell (Mond and Amson, 1928; Fennand Cobb, 1934), prompted a fundamental rethinking of the theory.

ORIGIN OF THE CHANNEL

Faced with the need to explain the potassium-permeability issue, Boyleand Conway (1941) proposed an elegant solution: the potassium chan-nel. Since the hydrated potassium ion was known to be smaller than thehydrated sodium ion, 3.8 Å vs. 5 Å, Boyle and Conway proposed trans-membrane channels of critical size—large enough to pass potassium andits shell of associated water, but small enough to exclude sodium with itsshell. The membrane was effectively a sieve that passed small ions, butexcluded larger ones.

The Boyle-Conway atomic sieve theory was attractive in that it couldalso account for several known features of cell behavior without too muchdifficulty. It explained the accumulation of potassium inside the cell asan attraction to the cell’s negatively charged proteins (a so-called Donnaneffect). It explained the cell potential as arising from a charge separationacross the membrane (a capacitive effect). And it accounted for thechanges of cell volume that could be induced by changes of external po-tassium concentration (an osmotic effect). The sieve theory seemed toexplain so much in a coherent manner that it was immediately grantedan exalted status.

But another problem cropped up, perhaps even more serious than thefirst. The membrane turned out to be permeable also to sodium (Fig.1.1). The advent of radioactive sodium made it possible to trace the

Figure 1.1. Atomic sieve

theory. The size of the

channel was postulated to be

critical for passing potassium

and blocking sodium.

Observed passage of sodium

compromised the theory.

8

path of sodium ions, and a cadre of investigators promptly found thatsodium did in fact cross the cell boundary (Cohn and Cohn, 1939;Heppel, 1939, 1940; Brooks, 1940; Steinbach, 1940). This finding cre-ated a problem because the hydrated sodium ion was larger than the chan-nels postulated to accommodate potassium; sodium ions should have beenexcluded, but they were not. Thus, the atomic-sieve theory collapsed.

Collapse notwithstanding, the transmembrane-channel concept remainedappealing. One had to begin somewhere. A channel-based frameworkcould circumvent the sodium problem with separate channels for sodiumand potassium: if selectivity were based on some criterion other thansize, then distinct channels could suffice. A separate channel for sodiumcould then account for the observed leakage of sodium ions into the cell.

But leakage of sodium introduced yet another dilemma, of a differentnature. Sodium could now pass through the channel, flowing down itsconcentration gradient and accumulating inside the cell. How then couldintracellular sodium remain as low as it is?

ORIGIN OF THE PUMP

The solution was to pump it out. In more-or-less the same manner as asump-pump removes water that has leaked into your basement, a mem-brane pump was postulated to rid the cell of the sodium that would oth-erwise have accumulated inside.

The idea of a membrane pump actually originated before the sodiumproblem. It began at the turn of the last century with Overton, a promi-nent physiologist who had advanced the idea that the membrane wasmade of lipid. Realizing that some solutes could cross an otherwise im-permeable lipid membrane, Overton postulated a kind of secretory ac-tivity to handle these solutes. Through metabolic energy, the membranecould thus secrete, or pump, certain solutes into or out of the cell.

Debunking Myths 9

The pump concept resurfaced some forty years later (Dean, 1941), torespond specifically to the sodium-permeability problem. Dean did nothave a particular pumping mechanism in mind; in fact, the sodium-pumpwas put forth as the least objectionable of alternatives. Thus, Dean re-marked, “It is safer to assume that there is a pump of unknown mecha-nism which is doing work at a constant rate excreting sodium as fast as itdiffuses into the cell.” With this, the sodium pump (later, the Na/Kexchange pump) came decidedly into existence.

By the mid-twentieth century, then, the cell had acquired both channelsand pumps. With channels for potassium and sodium, along with pumpsto restore ion gradients lost through leakage, the cell’s electrophysiologyseemed firmly grounded. Figure 1.2 says it all.

Figure 1.2. The sodium

pump, adapted from a drawing

by Wallace Fenn (1953), one

of the field’s pioneers.

Na entry

Na transport

K penetration

Na pump

10

REFLECTIONS

Why have I dragged you through so lengthy a review? My purpose wasto demonstrate how channels and pumps arose. They came into beingnot because some a ler t sc ient i s t s tumbled upon them during agroundbreaking session at the electron microscope, but as ad hoc hypoth-eses needed to patch otherwise flagging theories. The channel arose whena putatively ion-impermeant membrane was found to pass potassium;the channel could pass potassium while properly excluding sodium andother larger hydrated ions. Then, sodium was found to enter the celland instead of reconsidering the channel concept, a second channel spe-cific for sodium was postulated. Sodium permeability also implied apersistent leak into the cell. To keep gradients from collapsing, a so-dium pump was postulated.

Once this hypothetical framework gained a foothold, it expanded bound-lessly. For the same reason that a sodium pump was needed, it becameevident that pumps for other solutes were needed as well. Virtually all ofthe cell’s solutes partition far out of electrochemical equilibrium (Stein,1990) and therefore need to be pumped. Hydrogen-ion pumps, calciumpumps, chloride pumps, and bicarbonate pumps to name a few, sooncame into being over and above the postulated sodium/potassium pumps.Yet, even at the height of this intense activity, Glynn and Karlish (1975)in their classic review had to reluctantly admit that notwithstanding anenormous thrust of experimental work on the subject, still no hypothesisexisted to explain how pumps pump.

The channel field exploded similarly. With the advent of the patch-clamptechnique (see below) in the late 1970s, investigators had gained the ca-pacity to study what appeared to be single ion channels. It seemed for atime that new channels were being identified practically monthly, manyof them apparently selective for a particular ion or solute. The numberof channels has risen to well over 100. Even water channels have comeinto being (Dempster et al., 1992). Elegant work was carried out to tryto understand how channels could achieve their vaunted selectivity (Hille,

Debunking Myths 11

1984). At least some channels, it appeared, could pass one ion or soluteselectively, while excluding most others.

Given the astonishing expansion of activity in these fields, could therebe any conceivable basis for doubt? Mustn’t the concepts of pumpingand channeling be as firmly grounded as any biological principle?

It is tempting to answer by placing side-by-side the key experiments origi-nally adduced to confirm pumping and channeling together with the pub-lished challenges of those experiments (Troshin, 1966; Hazlewood, 1979;Ling, 1984, 1992). I hesitate to recapitulate that debate because thechallenges are largely technical. Readers willing to invest time in acquir-ing familiarity with technical details are invited to consult these sourcesand consider whether the published concerns are valid or not.

Another approach is to consider evidence that could potentially lie inconflict with these concepts. Unlike mathematical theorems, scientifictheories cannot be proven. No matter how much evidence can be mar-shaled in support of a theory, it is always possible that some new piece ofevidence will be uncovered that does not fit, and if such evidence is bothsound and fundamental, the theory may require reconsideration. As weshall see in the next sections, certain basic questions concerning pumpsand channels have not yet been adequately dealt with.

CHANNELS REVISITED

The existence of single ion channels appeared to be confirmed by ground-breaking experiments using the patch-clamp technique. In this tech-nique the tip of a micropipette is positioned on the cell surface. Throughsuction, a patch of membrane is plucked from the cell and remains stuckonto the micropipette orifice (Fig. 1.3A). A steady bias voltage is placedacross the patch, and the resulting current flow through the patch ismeasured. This current is not continuous; it occurs as a train of discretepulses. Because the pulses appear to be quantal in size, each pulse is

12

+

Figure 1.3. “Single channel”

currents recorded in situations

depicted at left of each panel:

(A) after Tabcharani et al.

(1989); (B) after Sachs and

Qin (1993); (C) after Lev et

al. (1993); (D) after

Woodbury (1989). Note the

similarity of experimental

records, implying that the

discrete currents are not

necessarily related to features

specific to biological channels.

102.4 ms

4 pA

time (s)

-10

0

10

0 2 4 8

curren

t (p

A)

6

polyethyleneterephthalate filter

-

silicon rubber patch

membrane patch

0 40 80 120 160 200

10 p

A

time (ms)

+

- pure lipid bylayer

1 2 30time (s)

100

pS

A

B

C

D

cond

ucta

nce

Debunking Myths 13

assumed to correspond to the opening of a single ion channel.

This dazzling result has so revolutionized the field of membrane electro-physiology that the originators of the technique, Erwin Neher and BertSakmann, were awarded the Nobel Prize. The observation of discreteevents would seem to confirm beyond doubt that the ions flow throughdiscrete channels.

Results from the laboratory of Fred Sachs, on the other hand, make onewonder. Sachs found that when the patch of membrane was replaced bya patch of silicon rubber, the discrete currents did not disappear (Sachsand Qin, 1993); they remained essentially indistinguishable from thosemeasured when the membrane was present (Fig. 1.3B). Even more sur-prisingly, the silicon rubber sample showed ion-selectivity features es-sentially the same as the putative membrane channel.

A similarly troubling observation was made on polymer samples (Lev etal., 1993). Current flow through synthetic polymer filters was found tobe discrete, just as in silicon rubber (Fig. 1.3C). The filters also showedfeatures commonly ascribed to biological channels such as ion selectiv-ity, reversal potential, and gating. Yet, the sample was devoid of anyprotein or lipid.

In yet another set of experiments, channel-like behavior was observed inpure lipid-bilayer membranes (Woodbury, 1989). Following brief expo-sure to large concentrations of lipid vesicles ejected from a pipette tipapproximately 0.5 mm distant, these membranes showed typical chan-nel-like fluctuations (Fig. 1.3D). Conductance changed in ways usuallyconsidered to be indicative of reconstituted protein channels—includ-ing step conductance changes, flickering, ion selectivity, and inactiva-tion. But no channels were present; the membranes contained only lipid.

What are we to do with such observations? It is clear from these threestudies that the discrete currents previously taken to confirm the exist-ence of single biological channels seem to be general features of current

14

flow through small samples. The currents presumably arise from somecommon feature of these specimens that is yet to be determined, butevidently not from single channels since they are absent. The channelsmay exist—but the prime evidence on which their existence is based isless than conclusive.

Ironically, the silicon-rubber test had actually been carried out as a con-trol in the original patch clamp studies (Neher et al., 1978). The au-thors did sometimes note “behavior contrary“ to what was expected (p.223); but such behavior was dismissed as having arisen from irregulari-ties of the pipette tip. The possibility that small samples in general mightgive rise to channel-like behavior was apparently not considered.

Setting aside the above-mentioned concern, a second point to consideris the manner in which the channel achieves its specificity. Channelsexist for each one of the cell’s ions; additional channels exist for aminoacids, peptides, toxins, and sugars, most of these being otherwise unableto cross the lipid bilayer; and as I mentioned, there are also channels forwater. Thus, a plethora of channels exists, most engineered to be solute-specific. How is such specificity achieved?

To explain such exquisite specificity, models of some complexity haveevolved (Hille, 1984; 1992). One model contains 16 different transi-tion states, plus additional sub-states. Another contains 27 states. Mostmodels are sufficiently complex that the solution requires numerical meth-ods. Indeed, calculating the trajectory of a molecule diffusing through achannel during a 100-picosecond time window is the work of asupercomputer. Thus, models of daunting complexity are required forunderstanding the channels’ apparent selectivity. It is not a simple pro-cess.

The naive question nevertheless lingers: How is it that small solutes donot pass through large channels? To imagine how one or two small ionsmight be excluded from a large channel as a result of a distinctive elec-tric field distribution is not too difficult to envision. But the theory

Debunking Myths 15

implies that smaller solutes should be excluded as a class—otherwise,independent channels for these solutes would not be required. Thisenigma harks of the dog-door analogy (Fig. 1.4). Why bother adding acat door, a ferret door, a hamster door and a gerbil door when these smalleranimals could slip readily through the dog door? Must some kind ofrepellent be added for each smaller species?

The seriousness of the size problem is illustrated by considering the hy-drogen ion. The hydrogen ion is only about 0.5 Å in diameter (the hy-drated ion somewhat larger). The sodium-channel orifice is at least 3 - 5Å across, and channels specific for some of the larger solutes must have a

Figure 1.4. Dog-door

analogy. Door is large enough

to pass all smaller species.

16

minimum orifice on the order of 10 - 20 Å. How is it possible that a 20Å channel could exclude a diminutive hydrogen ion? It is as though atwo-foot sewer pipe that easily passes a beach ball could at the same timeexclude golf balls, as well as tennis balls, billiard balls, etc.

Arguably, the situation is not so black and white. Textbook depictionsof the channel as a hollow tube oversimplify the contemporary view ofthe channel as a convoluted pathway; and the process of selectivity isthought to rest not on size per se but on some complex interaction be-tween the solute’s electric field and structural features of the channel’sfilter (e.g., Doyle et al., 1998). Also, channel selectivity is not absolute(Hille, 1972). Nevertheless, the issue of passing only one or a few amonga field of numerous possible solutes including many smaller ones remainsto be dealt with in a systematic manner. And the issue of non-biologicalsamples producing single-channel currents certainly needs to be evalu-ated as well. What could all this imply?

PUMPS REVISITED

Like channels, pumps come in many varieties and most are solute-spe-cific. The number easily exceeds 50. The need for multiple pumps hasalready been dealt with: unless partitioning between the inside and out-side of the cell is in electrochemical equilibrium, pumping is required.Because so few solutes are in equilibrium, one or more pumps are neces-sary for each solute.

A question that arises is how the cell might pump a solute it has neverseen. Antibiotics, for example, remain in high concentration outside thebacterial cell but in low concentration inside. Maintaining the low in-tracellular concentration implies the need for a pump, and in fact, a tet-racycline pump for E. coli has been formally proposed (Hutchings, 1969).A similar situation applies for curare, the exotic arrow poison used ex-perimentally by biophysicists. Because curare partitioning in the muscle

Debunking Myths 17

cell does not conform to the Donnan equilibrium, a curare pump hasbeen proposed (Ehrenpries, 1967). To cope with substances it has neverseen, the cell appears to require pumps over and above those used on aregular basis—on reserve.

How is this possible? One option is for existing pumps to adapt them-selves to these new substances. But this seems illogical, for if they couldadapt so easily why would they have been selective to begin with? Analternative is for the cell to synthesize a new pump each time it encoun-ters a foreign substance. But this option faces the problem of limitedspace: Like the university parking lot, the membrane has just so manyspaces available for new pumps (and channels). Given chemists’ abilityto synthesize an endless variety of substances—10 million new chemicalsubstances have been added to the American Chemical Society’s list ofmolecules during the last quarter century alone (N. Y. Times, Feb 22,2000)—how could a membrane already crowded with pumps and chan-nels accommodate all that might eventually be required? Could a mem-brane of finite dimension accommodate a potentially infinite number ofpumps?

A second question is how the cell musters the energy required to powerall of its pumps. Where might all the ATP come from? Since ions andother solutes cross the membrane continually even in the resting state(in theory because of sporadic channel openings), pumps must run con-tinuously to counteract these leaks. Pumping does not come free. Thesodium pump alone has been estimated, on the basis of oxygen-consump-tion measurements, to consume 45 - 50% of all the cell’s energy supply(Whittam, 1961). Current textbooks estimate a range of 30 - 35%.

To test whether sufficient energy is available to power pumping, a well-known experiment was carried out long ago by Ling (1962). Ling fo-cussed on the sodium pump. The idea was to expose the cell to a cock-tail of metabolic poisons including iodoacetate and cyanide, and to de-prive it of oxygen—all of which would deplete the cell of its energy sup-ply and effectively pull the pump’s plug. If these pumps had been re-

18

sponsible for maintaining sodium and potassium gradients, the gradi-ents should soon have collapsed. But they did not. After some eighthours of poison exposure and oxygen deprivation, little or no change incellular potassium or sodium was measurable.

Ling went on to quantitate the problem. He computed the residual en-ergy—the maximum that could conceivably have been available to thecell following poisoning. This residual was compared to the energy re-quired to sustain the ion gradient, the latter calculable from the knownsodium-leak rate. Using the most generous of assumptions, a conserva-tive estimate gave an energy shortfall of 15 to 30 times (Ling, 1962).The pump energy needed to sustain the observed gradient, in other words,was concluded to be at least 15 to 30 times larger than the availableenergy supply.

This conclusion stirred a good deal of debate. The debate was high-lighted in a Science piece written by the now well-known science writerGina Kolata (1976), a seemingly balanced treatment that gave credenceto the arguments on both sides. Kolata cited the work of Jeffrey Freed-man and Christopher Miller who had challenged Ling’s conclusion aboutthe magnitude of the energy shortfall. Ling’s late-coming rebuttal (1997)is a compelling “must-read” that considers not only this specific issuebut also the process of science. The energy-shortfall claim was neverthe-less left to gather dust with the advent of pump-protein isolation—for-gotten by all but a modest cadre of researchers who have remained stead-fastly impressed by the arguments (cf. Tigyi et al., 1991).

In retrospect, any such niggling debate about the magnitude of the so-dium-pump-energy shortfall seems academic, for it is now known thatnumerous other pumps also require power. Over and above sodium andpotassium, the cell membrane contains pumps for calcium, chloride, mag-nesium, hydrogen, bicarbonate, as well as for amino acids, sugars, andother solutes. Still more pumps are contained in organelle membranesinside the cell: In order to sustain intra-organelle ion partitioning, or-ganelles such as the mitochondrion and endoplasmic reticulum contain

Debunking Myths 19

pumps similar to those contained in the surface membrane. Given leakrates that are characteristically proportional to surface area, we are notspeaking here of trivial numbers of pumps: Liver cell mitochondria con-tain 20 times the surface area of the liver cell membrane (Lehninger,1964), and the area of the muscle’s sarcoplasmic reticulum is roughly 50times that of the muscle cell membrane (Peachey, 1965). Membranes ofsuch organelles must therefore contain pumps in numbers far higher thanthose of the cell membrane—all requiring energy.

In sum, pumping faces obstacles of space and energy. The membrane’ssize is fixed but the number of pumps will inevitably continue to grow.At some stage the demand for space could exceed the supply, and whatthen? Pumping also requires energy. The Na/K pump alone is estimatedto consume an appreciable fraction of the cell’s energy supply, and thatpump is one of very many, including those in internal membranes. Howis the cell to cope with the associated energy requirement?

COULD CHANNEL AND PUMP PROTEINS PLAY ANOTHER ROLE?

The sections above have outlined certain obstacles faced by the pump –channel paradigm. But proteins exhibiting pump-like or channel-likebehavior have been isolated and their existence needs to be explained. Ifnot specifically for pumping and channeling, why might they be present?

One plausible hypothesis is that they exist for some different purpose.Given their superficial location, “pump” and “channel” proteins couldtrigger a chain of events leading ultimately to action in an intracellulartarget. Conformational changes are known to occur not only in pumpand receptor proteins but in channel proteins as well (Kolberg, 1994). Ifsuch changes were to propagate inward, the pump or channel proteinwould effectively play the role of a receptor.

A scenario of this sort need not contradict the proteins’ classical “pump-

20

ing” or “channeling” action: As the conformational change proceeds,any bound ion will shift in space along with the protein (Fig. 1.5). If thecharge shifts against the voltage gradient, the result will be interpretedas pumping; if it shifts with the gradient the ion will be presumed tohave passed through a channel. Thus, pumping or channeling would bea natural inference, even though the protein’s functional role is as nei-ther a pump nor a channel.

A good example to illustrate this kind of behavior is colicin Ia, a toxinmolecule that insinuates into bacterial membranes. As it does, it isthought to create a channel that allows ions to pass, thereby collapsingion gradients and killing the cell. The protein’s action is associated withsubstantial conformational change (Fig. 1.6); some 50 amino acids flipfrom one side of the membrane to the other—along with their boundions. Such charge shift would constitute a pulse of current similar to thecurrents depicted in Figure 1.3; hence, channeling is implicit. Whethersuch “channel” current mediates the protein’s toxic function, however, isless certain. Toxicity could as well lie in the protein’s altered configura-tion, inhibiting some vital process through interaction with cell proteins.

Another example is rhodopsin. Rhodopsin is a retinal receptor moleculethat undergoes conformational change in order to signal the presence oflight. Rhodopsin exists in another form called bacteriorhodopsin. Alsodriven by absorbed light energy, bacteriorhodopsin can translocate pro-tons across the bacterial cell membrane. Thus, rhodopsin is a light-drivenreceptor while bacteriorhodopsin is presumed to be a light-driven pump.Again, the charge movement observed in bacteriorhodopsin may not nec-essarily be the main event—the protein could function as a receptor of

Figure 1.5. Translocation of

charges across the membrane

will be registered as current

pulses.

+ + ++ + ++ +

_ _ _ _ _ _

+

+ + + + ++

+ +

_ _ _ _ __+

Debunking Myths 21

light just as rhodopsin does in the retina, triggering a response throughconformational change (Lewis et al., 1996). As with the channeling ac-tion of colicin Ia, the pumping action of bacteriorhodopsin might thenbe an incidental byproduct, and not necessarily the primary event.

Whether the observed pumping or channeling seen in such moleculescould be of functional significance would depend on their magnitude: Ifthe “pump” molecule translocates relatively few ions, its contribution tocellular ion separation would be small. And if the “channel” fails tocarry the lion’s share of ion traffic through the membrane, it too wouldplay little or no role in partitioning. Even though experimentally ob-servable, pumping and channeling by these proteins would then remainfunctionally insignificant—much like the heat generated by a light-bulb.

It seems, then, that this section’s conundrum may be resolvable. Althoughtests imply that the proteins under consideration can “pump” or “chan-nel,” such processes may be incidental to the proteins’ main functionalrole as receptors. Receptor proteins are often closely linked to pumpand channel proteins in order to “modulate” their activity; here they mergeinto a single unit whose contributions to ion partitioning may be en-tirely secondary.

Figure 1.6. Channel protein

that opens by flipping from

one side of the membrane to

the other. After Slatin et al.

(1994).

NH2

NH2COOH COOH

lipid bilayer

22

CONCLUSION

Some bold leaps have been taken in this chapter. We began by grantingourselves license to explore two basic elements of modern cell biology—channels and pumps. As we reviewed their origin, we found that theyarose as postulates, put forth to rescue attractive theories that would oth-erwise have collapsed.

The framework surrounding those postulates then grew in complexity.Channels and pumps multiplied rampantly in number and their featuresgrew devilishly intricate. In order to achieve selectivity, the channelneeded many states and sub-states; and the pump was required to handlesubstances it had never before seen. These complexities hinted that some-thing could well be amiss.

Some things were indeed amiss, or at least questionable. For the chan-nels it was the lead provided by the patch-clamp experiments. Thoseexperiments had been taken as proof of the existence of discrete biologi-cal channels, but that evidence has been thrown into doubt by the dem-onstration that similar results could be obtained when channels wereabsent. Also considered was the selectivity issue. It was not clear howthe channel could pass one solute primarily, while systematically exclud-ing others of the set—particularly its smaller members (the dog-doorproblem).

Questions were also raised about pumps. One issue is the means by whicha membrane of finite surface area could accommodate a continually grow-ing number of pumps (and channels)—what happens when space runsout? A second issue is the nettlesome one of energy-balance: if the cell’senergy supply is marginally adequate to handle sodium pumping, whatresources are available to power all of the rest of the many pumps?

Although this chapter’s goal was to begin constructing a functional edi-fice, the challenge of finding solid foundational ground has not yet been

Debunking Myths 23

met. The foundation remains uncertain. Nor can the soundness of sub-structural layers be presumed, for the pump and channel questions seemprofound enough to hint that the problems could originate more deeply.

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