CALCIUM IONS

by David R. Zimmerman

surge of excitement is coursing "' • t h rough the small, scattered

community of researchers who have long probed the essential role in muscle activity of the chemical element calcium. After years of frustration, these researchers believe that they are about to close a critical gap in their knowledge: They are answering—rapidly now—the question of how a muscle cell is trig™ gered to twitch, or contract.

More broadly, the researchers believe they are answering the question about the way all muscular movement is ac­tivated, from heartbeats to handstands to the closing of the lowliest bivalve's shell Half of an animal's body mass is muscle; and the present discoveries promise significant new understanding of its function and control.

Anatomists and cell physiologists, electron microscopists, biochemists, and biophysicists have been scratching with fascination for more than two decades at the problem of how muscle works. The swift pace of these researchers ' present progress can be gauged by a statement published as recently as Au­gust 1988 in the staid pages of the Fed­eration of European Biochemical Societies Letters: "The enigma of excitation-con­traction coupling in skeletal muscle . . . remains one of the major problems of muscle physiology."

This enigma now is largely, albeit not whoEy, solved. New discoveries and new understanding of a long chain of earlier findings are throwing light not only on the firing of all voluntary, or skeletal, muscle cells but also on the regulation of involuntary muscle, including heart and smooth muscle and perhaps a wide range of other cell types as well.

The p r e g n a n t and no doubt deli­riously anticipatory plaint in the Euro­pean biochemists' Letters in fact was written on this side of the Atlantic, at

the University of Pennsylvania School of Medicine in Philadelphia, a major center for the study of calcium's role in muscle physiology. Senior author Andrew P. Somlyo, a cardiologist-cwm-physiolo-gist, developed key pieces of the hard­ware, and some exacting experimental standards, that led to the present suc­cess. A co-author at Pennsylvania, phys­

iologist Stephen M. Baylor, now works on major questions that remain. (Som­lyo and his wife and co-investigator, physiologist Avril V. Somlyo, recently moved their laboratory to the University of Virginia in Charlottesville.)

Still another University of Pennsyl­vania investigator, cell physiologist Clara A. Franzini-Armstrong, played a signif-

14 MOSAIC Volume 20 Number 3 Fall 1989

The question of what makes a muscle twitch rapidly became

icant role in defining the questions in the 1960s. She is now participating en­thusiastically in its denouement.

Coupling

The p h r a s e excitat ion-contract ion coupling (E-C coupling), broadly speak­ing, describes the physiological process through which a muscle cell is electri­cally depolarized and then induced to contract. Scientists have understood the excitation half of the process fairly well: An electrical impulse, or action poten­tial, is evoked by a chemical neurotrans­mitter released by an adjacent nerve cell. The impulse then travels along a skeletal muscle cell's outer membrane.

Scientists initially believed that the de­polarization of the cell's outer mem­brane directly triggered muscle con­traction. However, experiments soon showed that, at least in skeletal muscle, this is not so. Rather, the electrical mi­crocurrents turn inward and flow into narrow tunnels that penetrate the mus­cle cells at 90-degree angles, like the side passages of a coal mine. These tunnels are called transverse tubules, or T-tu-bules. They are organized in networks and are quite abundant; Franzini-Arm­strong estimates that a single 40-milli-meter-long muscle cell in a frog's leg contains five or six million such tunnels.

It was at this point, as the electrical impulse travels inward along the myriad T-tubule walls, that matters became— and to an extent remain—murky.

Jump, now, across two gaps, one physical and one conceptual, to the end of the contraction part of the E-C cou­pling process. Researchers have known for about two decades that a muscle cell twitches—contracts-—when calcium re­moves a chemical chock, thereby allow­ing the force-producing fibrils of the proteins actin and myosin to interact. A pulse of calcium in the cytoplasm sur­

rounding these fibrils is the immediate stimulus. Calcium thus is the biochem­ical trigger of muscle contraction.

Researchers once thought that this calcium crossed the cell membrane and entered the cell from the calcium-rich extracellular fluid. This notion,. Franzini-Armstrong says, was dispelled once and for all by University of Pennsylvania bi­ologist Annmarie Weber and others, who showed that muscle cells contain a rich membrane system that can trap and hold large amounts of calcium, reducing its concentration In the cytoplasm to very low levels.

A powerful pump

Subsequent to this discovery, Andrew and Avril Somlyo developed instru­ments to track calcium within cells. Us­ing calcium dyes , the Somlyos can disclose calcium's instantaneous pas­sage across cell membranes; its fleet transients, or movements, within the cy­toplasm; and its re-accumulation within Intracellular depots. (See "To track a swift ion,"accompanying this article.) These studies confirm that the calcium trig­gering the contraction of a muscle cell comes from neighboring storage areas within that cell.

The key depots for the calcium used in muscle contraction are organelles In muscle cells called the sarcoplasmic re-ticula, or SR. (In other cell types these organelles are called endoplasmic retic-ula.) The complex, irregular shape of the SR belies the organelle's simple function. The sarcoplasmic reticulum, as biophy-sicist Kevin P. Campbell of the Univer­sity of Iowa College of Medicine in Iowa City explains, is a "balloon filled with

Suction Solution

Pump

MOSAIC Volume 20 Number 3 Fall 1989 15

Zimmerman From K.P. Roos et al., Proc. Intematl. Soc. Photo-Optical Instr.

calcium." But the SR is also a powerful pump—a vacuum cleaner, as it were, that sucks calcium safely back into stor­age, enabling the muscle fibrils to relax. "A wall-to-wall calcium pump over its entire length" is the way Stephen Baylor describes this organelle's membrane. The Somlyos can delineate the calcium stored inside these organelles. They have also shown that the SR releases calcium when the muscle contracts.

In related experiments several years ago, biochemist Gerhard Meissner and his associates at the University of North Carolina School of Medicine in Chapel Hill demonstrated that when isolated SR vesicles, or sacs, were properly stimu­lated with chemical messengers—par­t icular ly calcium and a d e n o s i n e triphosphate, or ATP—the calcium within flowed rapidly out. This efflux, Meiss­ner says, was comparable to the out­pouring of calcium from the SR during E-C coupling. Other researchers have re­ported like findings.

Excitation-contraction coupling is ex­tremely rapid. In skeletal muscle, the whole process—from stored calcium's release to twitch to calcium's recall and

Zimmerman is a freelance science writer based in New York City. His most recent contri­bution to Mosaic was "The Best Defense" in Volume 19 Number 1 Spring 1988.

Coming to terms with a killer

Ubiquity and toxicity—these traits of calcium are emerging as key reasons for its many regulatory roles within living cells. A third reason for calcium's newly recognized prominence is its avidity, its predilection for binding to and changing other molecular substrates.

Calcium at high concentrations is ubiquitous in ocean water, according to Cornell University cell biologist Robert B. Silver; the element may have been comparably concentrated in the primal soup. The extracellular fluid of mam­mals, too, contains a similar high concentration of calcium.

Some cellular organelles store comparably high concentrations of the metal. However, in the cytoplasm and other organelles, calcium molecules are spar­ser: about a thousandth to a ten-thousandth of their extracellular levels.

Calcium is highly toxic and in cells must be tightly controlled. Calcium can kill ceils in many ways. It turns on enzyme systems that break down parts of cells, Franzini-Armstrong says. Calcium will bind phosphorus to form calcium phosphate, a hard stuff that is incompatible with intracellular func­tion. So will carbon. Yet phosphorus is a key element in energy metabolism within living cells; carbon is also essential. So intracellular calcium must be kept scarce, to prevent these precipitates from forming. Too much loose calcium would, as it were, turn cells to bone.

Early in their evolution cells developed powerful pumps that forced calcium out through molecular pores in their outer membranes, thereby evading destruction. Cells also sequester calcium securely inside organelles such as the sarcoplasmic reticulum. Calcium control consumes much of a cell's en­ergy—about 15 percent in muscle cells, says physiologist Stephen Baylor of the University of Pennsylvania.

"Cells control calcium because it is so deadly to them," adds University of Iowa biophysicist Kevin Campbell. "By controlling calcium so well, millions of millions of years ago, cells began to use it as a messenger. Very low amounts of calcium could be brought into the cell to trigger a cellular response."

Ultimately, of course, it is cells (and organisms), which are alive, that regulate their functions; not calcium, which is inert. Seen from this perspec­tive, says University of Massachusetts botanist Peter Hepler, "the cell was in a position to exploit the enormous calcium gradient across the membrane for signal purposes, for muscle contraction, and ever-so-many other things. Cells developed a relatively efficient way to use as an agent what is—and would be—a poison to the cell!" •

16 MOSAIC Volume 20 Number 3 Fall 1989

the fibrils' relaxation—• takes about 0.2 second. In some extremely fast muscle fibers of certain fishes the whole process is completed in just 0.01 second.

The bridging question

This portrayal of E-C coupling of course begs the questions of how and where calcium is released from the SR and how electrical excitation of the cell membrane and T-tubules is transformed into cal­cium release from the SR and thence into muscle contraction. Pennsylvania's Clara Franzini-Armstrong approached these problems by studying electron micro­graphs she made of muscle cell cut into slices. She saw the calcium-filled bulges of the SR, whose outermost membranes lay close to and parallel with—-but did not touch—the walls of the T-tubules.

Furthermore, by squinting into her microscope's eyepiece and staring hard at her micrographs, Franzini-Armstrong could dimly discern linear arrays of bumplike projections on the SR-mem-brane side of the gap. These projections reminded her of rows of caterpillar feet seen from, below, and so she named them the "SR feet." The time was the late 1960s. As yet there was no way to study the feet carefully.

The gap between the facing mem­branes could be measured at that time, h o w e v e r , at about ten nanomete rs . Franzini-Armstrong suspected that this gap was the hyphen in excitation-con­traction, the junction between the T~tub-ule a n d the excitat ion phase of E-C coupling on the one hand, and the SR and ensuing contraction phase on the other. Still, she could not answer the question of how this gap is bridged each time any muscle moves.

A prescient speculation on this ques­tion came in the early 1970s from Yale physiologist W. Knox Chandler and his protege Martin F. Schneider. The two men based their proposition on Schnei­der's careful studies of electrophysiolog­ical charge movements during T-tubule depolarization. "Suppose that [electri­cally] charged groups of molecules were located in the membranes of the T-[tub­ule] system," Schneider and Chandler wrote in Nature in 1973, and suppose, too, " t ha t their displacement consti­tuted a step in E-C coupling. If the groups were attached to long molecules which extended to the adjacent 'foot' proteins oftheSR . . . they would provide a means by which the [electrical] potential across the wall of the T-[tubule] system could

Nanometer freeze frame. In a snap-freeze chamber, high concentrations of calcium (dark grey spheres) and potassium (light grey streaks) in rat atrial cardiac cells are revealed in computer-generated x-ray map.

be sensed by the SR. In fact, movement of the [electrically charged] molecules could directly regulate the release of cal­cium [from the SR]."

The means for direct confirmation of the Yale scientists/ hypothesis did not exist at the time. Progress, rather, re­flected slow, hard-fought improvements in methods and instrumentation at a number of laboratories. In Franzini-Armstrong's University of Pennsylvania laboratory this progress came In the form of ever more incisive methods for freez-

ing, fracturing, shadowing, and Imag­ing the critical junctional gap. As these methods improved, the SR feet came ever more clearly into focus, facilitating new interpretations of their physiologic role. To assist her own thought process and to explain it to others, Franzini-Arm-strong began to fashion models out of wood and plastic that represented the opposing T-tubule and SR membranes, complete with feet.

Franzini-Armstrong also began to look for SR and SR feet in a wide variety of organisms, from fish, scallops, and even simpler sea creatures to mammals . Wherever she found muscle, she found SR and their feet. This ubiquity has led Franzini-Armstrong to postulate that SR feet—and one can assume also their function—"are one of the most con­served structural features of muscle fi­bers, occurring with the same basic disposition and spacing at the junctions between internal and surface mem­branes of all types of muscle fibers."

"The junction in scallops looks very much like the junction in vertebrates," the Pennsylvania cell physiologist adds.

By the early 1980s researchers could discern the feet extending clear through the sarcoplasmic reticulum, like a line of pegs in a child's pegboard toy. Further­more, they could see that each individ­ual foot was composed of four large, equal subunits, like lucky clover leaves, with what appeared to be an indentation or opening where the subunits met at the center. The large size of the foot and of each of its four subunits soon proved to be important clues.

Calcium channels

The progress thus far largely reflected visual and electrophysiological analyses of intact muscle cells. Further analysis, at the molecular level, now required ex­traction of the junctional SR and T-tubule membranes from whole muscle cells and their dissolution by detergents. The re­sultant mush then is purified by cen-trifugation, so that individual molecular components can be segregated accord­ing to their molecular weight.

These procedures were applied to SR membranes almost simultaneously in several laboratories, including Gerhard Melssner's in Chapel Hill, Campbell's In Iowa City, and those of researchers Sid­ney Fleischer at Vanderbilt University in Nashville, Tennessee, and Anthony Caswell at the University of Miami. The researchers' efforts yielded a surprise: a

MOSAIC Volume 20 Number 3 Fall 1989 17

huge macromolecule, each of whose four Yale's W. Knox Campbell says. idly release calcium when treated with subunits had a molecular weight of about In one study, biochemist Meissner and physiologic messengers and modulators 400,000 daltons. The sheer size of the his co-workers studded a thin fatty ar- such as ATP. The group now showed that macr,omolecule seemed to invite further tificial cell membrane with these large calcium would pass rapidly through the study. "It's probably the largest mem- proteins. He and his associates already artificial membranes in a similar way. brane protein that's ever been found," had shown that isolated SR vesicles rap- Since the fatty membranes themselves

Calcium's myriad roles "Calcium is the most dynamic second messenger known;

no other intracellular signaling pathway fluctuates so rap­idly or is the subject of such intense and widespread in­terest," biologist Roger Y. Tsien of the University of California at Berkeley and his protege, Martin Poenie, re­cently declared.

Here are a few new and surprising biological regulatory roles for calcium:

Killer T-cells' accomplice: Killer T-cells are a type of white blood cell, in the immune system. When a killer T-cell attacks a virus-infected cell or cancer cell that has been targeted immunologically for destruction, the level of calcium within the T-cell rises dramatically. The calcium apparently comes from outside the cell. Given calcium's toxicity, one might guess that the killer cells fulfill their role by injecting it into their targets. But this apparently is not the case, reports developmental biologist Poenie, who recently started a laboratory at the University of Texas at Austin.

Poenie uses fura-2, a fluorescent dye developed by Tsien, which clearly depicts calcium's distribution and concentration in the T-cell during the ten minutes in which that cell approaches, grapples with, and dispatches a tar­get ceil. The highest calcium levels, he found, are in the far side of the T-cell, away from its area of contact with the target. This finding, which Poenie calls counter-in­tuitive and still in need of confirmation, suggests to him that calcium plays an essential though rearguard role in T-cells' attack. "A killer T-cell only hits one target cell at a time," Poenie says. "It doesn't shoot its cannons all at once."

Poenie hypothesizes that the calcium may help aim the

T-cell from the rear, since the buildup of calcium "fore­shadows" and "predicts" which of several adjacent target cells will be hit. The T-cell "goes through contortions," he says. "There is a lot going on inside it. Some internal rearrangements are taking place."

Possibly, Poenie speculates, the calcium concentration at the far side of the T-ceil creates a contractile wave that thrusts its leading edge forward and then onto and around the target cell, engulfing it for the kill.

Pacemaker for plant cell mitosis: Botanist Peter K. Hep-ler at the University of Massachusetts in Amherst is de­fining a key regulatory role for calcium in plant growth. His subjects are stamen hair cells of the wandering jew (Tradescantia). When Hepier reduces the amount of extra­cellular calcium available to the cells during mitosis, the ceils take longer to divide. Calcium clearly seems to reg­ulate this essential and universal function. Hepier believes that he is now "right at the edge" of defining one of what may be many ways in which calcium controls plant cell functions, as it is already known to do in animals.

Using experimental, crude dyes that bind calcium in plant cells without thoroughly disrupting the cells' func­tions, Hepier can demonstrate a sudden inflow of calcium ions at anaphase. (Anaphase is the mitotic stage in which the paired chromosomes at the parent cell's midline sep­arate and move toward opposite poles, preparatory to the parent cell's division into two daughter cells.) Hepier in­itially thought that calcium triggered anaphase. But closer study over several years has led him to modify this view: The calcium pulse can be shown to occur just after the paired chromosomes separate.

The chromosomes' movement to the poles, which in

18 MOSAIC Volume 20 Number 3 Fall 1989

are otherwise impervious to calcium, this finding was fair indication that the huge protein molecules were functioning as calcium release channels. The protein molecules, the researchers subsequently learned, tend to insert themselves into

the membrane in such a way that their channels will allow the efflux of calcium. ''[We were] measuring calcium ion cur­rents passing through an aqueous pore formed by an open channel/ ' Gerhard Meissner explains.

A second line of evidence linking the macromolecules to calcium flow devel­oped from experiments that employed an exotic plant poison-—a "most horrible agent / ' Pennsylvania's Clara Franzini-Armstrong calls it-—named ryanodine.

Tradescantia normally takes 20 minutes, seems to be facil­itated by a brief, temporary breakdown of strawlike mo­lecular structures called microtubules. These structures fill the space that the chromosomes must traverse between the cell's midline and its poles. Hepler had already dem­onstrated that calcium breaks down microtubules. His re­cent carefully timed observations now show calcium rising just after anaphase, as the chromosomes move toward the poles, and dropping back to baseline after they pass through. "One has reason to suspect that calcium is the intracellular regulator of microtubule formation," he states.

Mitotic trigger in animal embryogeeesis: Biologists long have suspected that calcium triggers the division of animal cells. Cornell University cell biologist Robert B. Silver, working at the Marine Biological Laboratory in Woods Hole, Massachusetts, says he now has directly confirmed this suspicion in the eggs of sand dollars. He chose these eggs for study because of their near-crystalline clarity.

Silver microinjected one of the two cells of an early (blastomere) sand-dollar embryo with a buffering agent that binds calcium. That cell stopped dividing, while its uninjected twin continued on. Injecting the stalled cell with calcium restarted its mitotic clock.

"These findings," Silver declares, "demonstrate that calcium ion transients are used to coordinate and syn­chronize the biochemical pathways required for mitosis. In addition, the data support the existence of a cell-cycle clock that in turn coordinates the biochemical pathways and the cell's receptivity to these calcium pulses."

Regulator of genetic expression: The commonplace view has held that transcription of a gene is free of somatic constraints. Molecular endocrinologist Bruce A. White at

the University of Connecticut Health Center in Farming-ton presents a different view. White says that a current hot topic in science is the regulation of genes, and that calcium is one of the regulators. "It is clear now," White declares, "that a lot of genes are regulated by second-messenger systems."

One gene regulated by calcium produces prolactin in cultures of rat pituitary cells; White and his mentor, phys­iologist F. Carter Bancroft of the Mount Sinai School of Medicine in New York City, were the first to demonstrate this, several years ago. They since have shown that, sur­prisingly, calcium exerts control early in the DNA-to-RNA-to-protein sequence of genetic events, apparently when DNA is transcribed into messenger RNA. By manipulating calcium levels in their cell cultures, White and Bancroft obtained 10-, 50-, even 200-fold increases in prolactin mes­senger RNA (rrtRNA), and thus in prolactin synthesis. Other mRNAs were not affected.

In recent studies Bancroft has shown that the calcium-responsive control unit of the prolactin gene lies close to the gene's 5' (transcriptional front) end, ahead of the tran­scriptional initiation site. This location, White says, argues for calcium's role as a transcriptional regulator, although more recent studies in his laboratory indicate that it may play a post-transcriptional regulatory role as well.

Why is calcium playing these roles? White says that in rats the hypothalamus down-regulates the hormone pro­lactin, and he speculates that calcium may up-regulate this hormone to keep it within normal physiological bounds. Scientists are just beginning to understand calcium's role in genetic regulation, White adds; two other unrelated genes now have been found to be regulated by it. •

MOSAIC Volume 20 Number 3 Fall 1989 19

This poison is extracted from South. American shrubs of that name. Ryano­dine protects these plants by paralyzing and killing leaf-eating insects. The poi­son also causes irreversible contraction, or spasm., in vertebrate muscle cells. It does so, Iowa City's Kevin Campbell and Chapel Hill's Gerhara MeC ,-r.er explain, by locking intracellular calcium, channels in an open position. Muscit fibrils thus are continually bathed in calcium ^rr1

therefore cannot relax. Ryanodine ~poi™ soned cells die within minutes.

In experiments, Campbell, Meissner, and others showed that ryanodine binds avidly to the large molecule that had been purified from the sarcoplasmic reticu­lum. This finding suggested that the ryanodine receptor molecule, as it came to be called, functions as a channel through which calcium in the sarco­plasmic reticulum is released into the cy­toplasm and adjacent muscle fibrils.

The circle of proof was closing. "The [SR] feet, the ryanodine receptor, and the calcium, ion release channel all seem to

To track a swift ion

A trace of calcium traverses a cell's cytoplasm, triggers a physiological event, and is gone in a trice. Finding and recording these calcium transients, or movements, and tracing their extra- and intracellular sources and targets has presented a forbidding technological challenge. Dif­fusible calcium, unlike solid tooth and bone, thus far can­not be visualized directly in biological systems.

A major research task, therefore, has been to create molecular tags that will bind to and reveal calcium without distorting its natural movement. At the same time, re­searchers have worked to develop instruments that image and record these markers, or, alternatively, that collect and analyze the atomic emanations that occur when cal­cium is ionized by high-energy sources.

As the distance to these goals has diminished in recent years, scientists have turned their attention to computers to create more sophisticated and detailed models of cal­cium's movements and physiological roles. Some of the first such findings, from a new facility sponsored by the National Science Foundation, are starting to appear.

The pace of progress can be judged by the fact that until a few years ago some investigators believed that calcium in smooth muscle and nonmuscle cells was stored in the mitochondria. The reason for that belief, explains Uni­versity of Virginia physiologist Andrew P. Somlyo, was that when cells were fixed and stained for calcium the calcium appeared microscopically in these organelles.

Instruments developed by Somlyo and his wife and co­worker, Avril V. Somlyo, have wholly shattered this view: Calcium collects in the mitochondria only when cells are injured or dying. In living muscle cells the sarcoplasmic reticulum, and homologous endoplasmic reticulum in other types of cell, are the major calcium depots.

To catch the fleet calcium atoms and ions in mid-tran­sient, Avril Somlyo developed a snap-freeze chamber. In it a kind of supercooled Freon popsicle freezes the outer five to ten micrometers of a living cell poised on an electron microscope's stage. The freezing takes only a few thou­sandths of a second, abruptly stopping all movement in the cytoplasm.

The electron microscopes with which these specimens are analyzed are equipped with mass spectroscopic probes.

Andrew Somlyo, who refined this method for use in bio­logical studies, can focus a high-energy beam on a patch of frozen cytoplasm or an organelle, with a fine resolution of ten nanometers. He obtains an instant readout of its concentrations of calcium, potassium, phosphorus, and other ions. Researchers have used this method, called elec­tron probe microanalysis, to map the intracellular distri­bution of calcium. The method, Andrew Somlyo says graciously, is "the physicist's gift to biologists."

Snap-freezing a cell stops its activity. The Somlyos and their colleagues also wanted to see calcium as it moved through cells.

The first such view came in the 1960s, when biologist Osamu Shimomura, now at the Marine Biological Labo­ratory in Woods Hole, Massachusetts, purified a sub­stance from the outer perimeter of jellyfish (genus Aequorea). This substance, called aequorin, luminesces as bright blue when exposed to calcium. Other researchers found that when they loaded living cells with aequorin, calcium's influx and intracellular transients were revealed.

Soon hundreds of laboratories were seeking aequorin. One early investigator, physiologist John R. Blinks of the Mayo Foundation in Rochester, Minnesota, has been the principal provider of aequorin for the last two decades. He and his colleagues dip-net thousands of the unpre­possessing, fried-egg-shaped invertebrates out of Pacific waters off the coast of Washington each summer and ex­tract the aequorin.

Neither Blinks nor anyone else has figured out why these jellyfish bioluminesce. They probably do not do it to attract each other, Blinks says, since they have no eyes. The best guess, in Biinks's view, may be the burglar-alarm hypothesis of James Morin of the University of California at Santa Barbara. When shrimp-like marine parasites nib­ble on the jellyfish, Morin proposes, the nibblers expose their host's aequorin to calcium in the sea water, signaling nearby fish to come dine on the parasites, thereby saving the jellyfish. "Mother Nature really was very kind to give us this tool," Blinks says.

Researches have conducted thousands of experiments with aequorin. Nonetheless, developmental biologist Mar­tin Poenie of the University of Texas at Austin finds the

20 MOSAIC Volume 20 Number 3 Fall 1989

he synonymous/ ' Meissner and his as­sociates wrote in the January 28, 1988, issue of Nature. Campbell, meanwhile, was comparing the physiological prop­erties of the ryanodine receptor and the pore of the calcium release channel. He also concluded that the receptor and the channel are one and the same. Early in 1989 Campbell and Franzini-Armstrong wrote: "It is assumed that the foot pro­tein represents the channel through which calcium is released during exci­tation-contraction coupling."

Recently, both the DNA sequence and the amino acid sequence of the foot pro­tein have been determined and pub­lished. In a report in the June 8, 1989 Nature molecular biologist Hiroshi Tak-eshima of Kyoto University and ten Jap­anese co-workers detailed the cloning and sequencing of the complementary DNA of rabbit skeletal muscle SR.

The result: a 5,037-amino-acid se­quence. The predicted structure, the re­searchers say, indicates that the calcium-release channel is in the C-terminal region

of the molecule. The rest of the molecule is the foot structure, which sticks out into and nearly spans the junctional gap.

Seeking the trigger

In the Campbell/Franzini-Armstrong model, then, the shamrock-like foot pro­tein is a calcium channel that opens up following electrical excitation of the T-tubule. Calcium spills briefly out into the cytoplasm, causing the nearby myo­fibrils to contract, after which the cal­c ium is p u m p e d back into the

MOSAIC Volume 20 Number 3 Fall 1989 21

substance to have significant drawbacks: Aequorin is not absorbed by cells and sometimes has to be microinjected into them. It is affected by magnesium ions in cells and other physiological factors. Moreover, since aequorin does not light up until it comes into contact with calcium, its presence in a cell cannot be confirmed in advance. Finally, aequorin does not always offer a good quantitative mea­sure of calcium.

Matters have significantly improved in the last several years, largely thanks to the labors of biologist Roger Y. Tsien of the University of California at Berkeley. As Poenie explains, Tsien has committed himself to the task of de­veloping new calcium markers, while encouraging others (Poenie included) to use them in particular studies.

The high point of Tsien's achievement thus far is a flu­orescent marker called fura-2, which binds strongly and selectively to calcium. This specificity is essential, Tsien says, for detecting the relatively few calcium ions that may be commingled in cytoplasm with far greater amounts of magnesium, sodium, and potassium.

Binding to calcium changes the fura-2 molecule's excitation spectrum under ultraviolet light to shorter wave­lengths. Researchers thus can measure calcium concen­tration as the difference between the fura-2 uv excitation spectrum and that of fura-2 plus calcium.

Fura-2 is brighter than aequorin. It is less likely, Poenie believes, to distort or disrupt cellular function, although Blinks says that the reverse may be true. Unlike aequorin, fura-2 can be incubated with—and will be absorbed into— ceils, sparing researchers the dicey task of injecting the dye into cells with microfine needles.

"Fura-2 has shown us that positive gradients are in­volved—and pulses, not just elevations," Poenie says. "We're learning a little bit about how a cell signals!" Still-better dyes, Tsien adds, are being tested.

The advent of supercomputers, meanwhile, has facili­tated a revolution in the study of calcium transients and other parameters of muscle contraction. A leading center for this work is the Mayo Foundation, where cell phys­iologist Stuart R. Taylor has built a high-speed digital im­aging system, dubbed CAMERA, for computer-assisted measurements of excitation-response activities. With

CAMERA Taylor can collect data more rapidly; analyze them more thoroughly; and present them, three-dimensionally, with greater clarity than ever before.

Previous imaging systems, which used video cameras, were limited to about 30 frames per second, Taylor says— too slow for accurate depiction of calcium stimulation's effect on individual segments of muscle fibrils. With CAMERA, which yields up to 5,000 images per second, Tay­lor can study these events as they occur. Using living, beating heart muscle of rats, Taylor says, he and his as­sociate Kenneth P. Roos can document "what goes on in microscopically small spaces in an individual cardiac mus­cle cell on a beat-to-beat basis."

The CAMERA system already has turned up some sur­prises. For example, the assumption has been that the individual contractile units, or sarcomeres, of a muscle cell fibril contract and then relax and reextend pretty much in unison. If they did not, the theory goes, the muscle cell would quickly tear itself to bits.

The CAMERA findings challenge this view. While the new imaging system confirmed that the calcium-induced "overall pattern of shortening and reextension was uniform in a given region of [a] cell, [and that] the onset of contraction was synchronous," there are "differences" in the behavior of adjacent sarcomeres, Taylor and his associates report. These sarcomeres varied in the distance they contracted by as much as one-third (31 percent), and differed, too, in the onset of reextension by as much as 25 milliseconds.

From these findings Taylor and his colleagues conclude "a certain degree of independence" between adjacent re­gions in a heart muscle cell, and that the links between regions are "weak or very elastic." These conclusions mean that a cell's overall twitch, which reflects the average be­havior of large numbers of its sarcomeres, "does not reflect the characteristics of individual sarcomeres."

Why this is so, Taylor says, still is unclear. However, the powerful new instrumentation that he and his co­workers are using promises new understanding of muscle contraction—and calcium's role in the process seems likely to grow rather than recede. "We've learned that calcium not only initiates contraction," Taylor says, "calcium and contraction also regulate one another interactively." •

0. 02 pM Co

^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^^^^^^m-

+ 2 mM ATP

sarcoplasmic reticula via a different route, and the muscle fibrils relax. This scheme, however, leaves one important question unanswered: How are the calcium chan­nels opened?

The answer is emerging from studies of the T-tubule side of the cytoplasmic gap. In 1973 Yale's Martin Schneider and W. Knox Chandler had proposed the ex­istence of long molecules that might ex-tend from the T-tubule membrane across to the SR feet. Soon after, Pennsylvania's Clara Franzini-Armstrong and other cell physiologists visualized these mole­cules. At first the researchers described them as "particles/' A decade of study has revealed these particles to be four-piece units, or tetrads, arrayed in lines

directly across the gap from the SR feet— virtually, if not actually, touching them.

The apposition of feet and tetrads sug­gested but did not wholly prove that the tetrads open the calcium channels. Again, the physiologists have had to defer to the molecular biologists. In Iowa City, Franzini-Armstrong's protege and col­laborator Kevin Campbell, among oth­ers , has isolated a large molecular complex from T-tubule membrane. Var­ious studies have suggested that this molecule is the basic unit of the tetrad. The molecule has four subuni ts , all binding sites for a calcium channe l blocking agent called dihydropyridine.

Discovery of the d ihydropyr idine binding sites immediately reactivated

speculation that the tetrad (the putative T-tubule membrane protein) opens dur­ing E-C coupling, allowing a tiny amount of extracellular calcium from inside the T-tubule to cross the gap and open up the larger channel inside the SR foot. When this larger channel opens, the cal­cium in the SR flows out and activates the muscle cell. This chain of events is called the calcium-mediated calcium re­lease hypothesis.

For some types of muscle cells, smooth muscle or heart muscle for example, this hypothesis may describe how the muscle fires, says Gerhard Meissner in Chapel Hill. But resul ts of an expe r imen t by University of Pennsylvania's Stephen Baylor and an English colleague, Stephen Hollingworth, which were published last year, suggest that calcium-mediated cal­cium release does not explain events in the voluntary skeletal muscle cells of frogs. Using a buffering agent that binds calcium ions in the cytoplasm, the two scientists showed that raising calcium levels in the gap probably does not trigger the SR chan­nels to open. If anything, calcium in the gap inhibits the release of other calcium from the sarcoplasmic reticulum.

If not calcium . . .

If calcium is not the trigger, then what is? Conceivably, Baylor says, some other chemical messenger crosses the gap to open the SR channels. An alternative, which is supported by recent electron micrographic studies, is Schneider and Chandler's 1973 hypothesis that projec­tions from the T-tubule side of the cyto-

Baylor, Studying the sarcoplasmic reticulum, !ta wall-to-wall calcium pump;

22 MOSAIC Volume 20 Number 3 Fall 1989

plasmic gap actually touch the SR feet, establishing a structural linkage that opens the channels, much as turning a faucet handle causes water to flow.

In this alternative view, which re­mains to be proved, the electrical exci­tation acts physically to change the shape of or move the T-tubule tetrads so that they interdigitate with and somehow open the SR channels. The Schneider/ Chandler hypothesis says, too, that the activity of the T-tubule projections responds functionally to the level of electrical excitation: The more the cell

membrane is depolarized, the stronger the reaction and hence the greater the amount of calcium released from the sar­coplasmic reticulum.

Viewed in this light, the T-tubule pro­jections become voltage sensors. As Pennsylvania's Franzini-Armstrong and several co-workers described these hy­pothetical events: "The initial step in excitation-contraction coupling is a volt­age-dependent rearrangement of mole­cules located in the T-tubule membrane, resulting in a detectable 'charge move-ment / Direct interaction between the

voltage sensor and the calcium release channel of the sarcoplasmic reticulum would . . . open . . . the latter. The T-tubule particles [tetrads/dihydropyri-dine receptors] are the most appropriate candidates [for] voltage sensors, and foot proteins in this scheme would perform the dual role of receiving the signal from the voltage sensor and being the release si tes/ '

This view of the tetrad's role gained strength late last year through the ex­periments of a Japanese-American team of researchers, including physiologist Kurt G. Beam at Colorado State Univer-sity in Boulder. The team used newborn mice of a lethally inbred strain. These mice are born paralyzed because their muscles are unable to contract. Al­though the mice possess foot proteins and other accoutrements of skeletal muscle function, their muscle cells are incapable of E-C coupling.

Beam and his associates isolated two structural mutations in the gene for the dihydropyridine receptor (the T-tubule tetrad) in these doomed mice and showed that the mutations paralyzed cells by failing to support E-C coupling. When the investigators introduced DNA for the normal gene into cultured mutant mus­cle cells, they restored E-c coupling in some cells—and also restored those cells' ability to respond to dihydropyridine and to function as calcium channels.

This experimental restoration of func­tion in mutant muscle cells "strongly supports the view that this . . . mole­cule in the T-tubule ... . is an essential component of E-c coupling, probably acting as a voltage sensor," Beam and his co-workers reported in the Novem­ber 10, 1988, issue of Nature. Biophysi-cist Kevin Campbell in Iowa City says: "We still don't know the mechanism to initiate release, that is, what's going to open the SR calcium channel." However, he adds, "at this stage we do know at least two of the players: the foot protein and the dihydropyridine receptor. This is a very exciting time!"

Concludes Pennsylvania's Franzini-Armstrong: "There is no experiment that confutes this hypothesis. In some point in your life you have to place your bets. I'm placing my bets on this!" •

The National Science Foundation contrib­utes to the support of the research discussed in this article principally through its Cell Biology and Instrumentation and Instru­ment Development Programs.

MOSAIC Volume 20 Number 3 Fall 1989 23