Wiring Up Essay, Research Paper

WHEN the commonplaces of one discipline are applied to an unrelated field,

they can prove curiously fruitful. In 1952 two British physiologists, Alan

Hodgkin and Andrew Huxley, managed just such a fruitful crossover, applying

textbook physics to living tissue. They were both later knighted, and

shared a Nobel prize in 1963. The experimental method they pioneered

remains fundamental to research into the behaviour of nerve cells.

As anyone who has ever had an electric shock knows, electricity has

powerful effects on living matter. Luigi Galvani found in 1771 that

electricity could make the muscles from frogs’ legs contract; soon

afterwards, physiologists came to suspect that all sensation and movement

depended upon electric pulses in nerve and muscle. But how does electricity

pass through living things?

By the time Dr Hodgkin and Dr Huxley (as they then were) came to these

questions, other researchers had discovered various things about nerve

cells. One of the most intriguing was that messages down nerves are as loud

when received as they were when transmitted–unlike messages sent down

cables, which attenuate with distance. Physiologists thought that this

active transmission had something to do with sudden and short-lived changes

in the electrical resistance of a nerve fibre’s outer membrane. The link

between transmission and changing resistance was the subject of decades of

increasingly intense speculation.

Progress was slow because the nerves were not, as the police put it,

assisting in the inquiries. Nerve fibres are made of axons, which are

hairlike protrusions that grow out of nerve cells. They are small and

delicate, unforgiving of rough treatment. The surges in the voltage across

the cell membrane, now called action potentials, are complex events lasting

only a couple of milliseconds. Difficulties with delicacy and speed often

thwarted the physiologists working on nerves before the second world war.

Another problem was the action potential’s uncompromising nature; it is

either present at full strength or absent altogether, never anything

in-between. Such all-or-nothing behaviour is a nightmare for scientists. It

means that varying the stimulus for an action potential causes no variation

in the response. It is from studying such variations that mechanisms are

normally revealed.

Throughout the 1930s Dr Hodgkin had been exploring electrical conduction in

nerves with some success, using many of the tools that he and his student

Dr Huxley were to exploit in their classic experiment. Many of these came

from America, where there were engineers skilled in producing the sensitive

electronic apparatus that was needed. In Cambridge, where Dr Hodgkin and Dr

Huxley had fellowships, physiologists had to build their own apparatus with

components bought from a local wireless shop. Another American import was

the object of study: giant nerve-fibres found in squid, as much as 40 times

larger than the largest vertebrate nerves, and thus far easier to dissect.

Despite these tools, though, the nature of the nerve proved elusive.

The difference between Dr Hodgkin’s pre- and post-war work is simple: the

war. Like other scientists, Dr Hodgkin and Dr Huxley broke off their

research when Britain declared war on Germany. Though train-ed as

physiologists, they were put to work in fields with a direct bearing on the

war effort: Dr Hodgkin worked on radar, Dr Huxley developed sights for

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Cole, who was another great influence on the Cambridge pair, and unlucky

not to share their Nobel laurels.

Clamped

An axon is a long tubular outgrowth from a cell, wrapped in a cell

membrane. One of the differences between the outside and the inside of the

cell is the concentration of various types of ion–atoms carrying electric

charge. To take one example, cells contain a high concentration of

positively charged potassium ions.

If the membrane becomes permeable to potassium ions, they will leak out of

the cell into the fluid outside. Force of numbers drives them from places

where they are concentrated to places where they are scarce. If the

membrane stops negatively charged ions joining the exodus, an electrical

potential, or voltage, quickly builds up across the membrane as positive

charge leaves the cell. Eventually that voltage becomes strong enough to

stop the flow of potassium. The electrical force encouraging the ions to

stay in the cell becomes as strong as the force driving them out.

The cell can quickly overturn this balance, though, by making its membrane

porous to other ions. These charged particles will flow to where they are

less common, just as potassium did, until a new balance between electricity

and concentration is struck. To the outside world, the movement of charge

shows up as a sudden change in the voltage across the membrane–an action

potential.

Dr Hodgkin and Dr Huxley realised that they could watch this process as it

happened by looking at ions flow across the membrane of a single nerve

fibre. They called the moving charge the “membrane current”, and set out to

measure it using Cole’s fancy electronic apparatus. They inserted two tiny

electrodes down the middle of the nerve. Since the electrodes could not be

allowed to touch, the wide-bore squid nerve-fibre was a godsend. Each

electrode was connected, through the membrane, to another in the fluid

outside the nerve. Currents in one of these pairs of electrodes were used

to “clamp” the membrane at a particular voltage. With the membrane

potential fixed by this first pair of electrodes, the second pair could be

used to measure the resulting membrane current.

Dr Hodgkin and Dr Huxley had found a way around the problems of

all-or-nothing action potentials. Like the good physicists the war had made

them, they had succeeded in controlling one variable–the potential–and

had thus won the freedom to explore how the other variable–the membrane

current–depended upon it.

The diagram summarises one set of results. It shows the currents that flow

at a spot on the membrane if the membrane potential is suddenly clamped at

a new value, higher than its resting value. Curve A is taken from a nerve

bathed in a fluid that is rich in sodium ions, as it would be in the body.

At first, charge flows into the cell; within a millisecond, it begins to

flow out again.

Richard Keynes, one of Dr Hodgkin’s students, had used radioactive isotopes

of sodium and potassium to show that the two elements moved in and out of

the nerve cell when it was stimulated. Armed with this information, Dr

Hodgkin and Dr Huxley could explain what was happening. Having realised

that changes in porosity lead to changes in voltage, they now argued that

changes in voltage lead to changes in porosity, as well.

Clamping the voltage at above its resting value makes the membrane porous

to positively charged sodium ions. They flood into the cell from outside,

where their concentration is high, bringing their positive charge with

them. That influx provides a sudden and transient inward current, seen in

curve B.

This leakiness to sodium is only transitory: the sodium current soon dies

away to nothing. Instead the membrane becomes porous to potassium. The flow

of potassium was isolated and measured by looking at a cell bathed in a

fluid containing no sodium ions: the result is shown in curve C. Potassium

flows out of the cell, carrying positive charge with it. Curves B and C

together add up to make curve A.

The overall effect is of a wave of current washing in and out of the cell.

The initial balance between the electric potential and the force driving

the ions across the membrane is disturbed. It swings first one way as

sodium pushes into the cell, then the other way as potassium rushes out. If

there was no clamp around, the current surge would make the voltage swing

wildly: that swing in voltage is the action potential. And it would change

the porosity of the membrane nearby.

Imagine the action potential running along an axon like a bead along a

thread. At the front edge of the bead, sodium is moving into the cell;

behind it, potassium is flowing out. In front of the bead, the oncoming

sodium current is increasing the voltage across the membrane; once the

voltage passes a certain level, the membrane becomes porous to sodium ions.

The action potential has arrived. Thus the ring of activity moves

forward–a pulse running along a nerve.

Dr Hodgkin and Dr Huxley had little time for generalisations, so they went

to remarkable lengths to develop their story.

They calculated the number of ions that crossed the membrane in an action

potential and showed that it agreed with Dr Keynes’s radioactive results.

They showed how the size of the action potential depends on the

concentration of sodium outside the nerve; the less sodium, the less the

force pushing sodium into the cell when the membrane becomes porous.

With data from a whole range of voltages, they used standard physics

calculations to work out what shape the action potential should have; their

answer matched measurements from living nerves almost exactly.

The finishing touch, ten years later, was similar in style: a physical

approach to the nerve. Peter Baker, who worked under Dr Hodgkin, found that

he could extrude a nerve-fibre’s innards, as one would squeeze toothpaste

from its tube. As long as the nerve-fibre is refilled with a mixture that

is rich in potassium but poor in sodium, it will go on to conduct as many

as 1m quite normal action potentials before it gives out. Dr Baker had

squeezed the life out of the nerve-fibre and turned it into an active

electrical wire. Cell biology had been reduced to textbook physics.

Back to discontinuity

Dr Hodgkin and Dr Huxley explained the action potential. They did not

manage to show the molecular mechanisms behind it. But those who came later

did, using similar techniques.

In 1976 two German physiologists, Erwin Neher and Bert Sakmann,

miniaturised the voltage clamp. Using a pipette with an opening only a few

millionths of a metre across, the voltage of a minute piece of membrane can

be clamped at any level, and the currents across it measured. The area is

so small that current can be seen switching on and off as a single hole in

the membrane opens and closes.

These holes–channels–turn out to be either closed or fully open: more

like switches than taps. As the voltage increases, the sodium channels

spend more of their time open. It is the combined effect of billions of

such channels that leads to the smooth curves seen by Dr Hodgkin and Dr

Huxley in a single nerve-fibre. As the channels open, the flow of sodium

boosts the potential even further, opening yet more. Then an automatic

shutting-mechanism comes into play. The potassium channels work on similar

principles, but more slowly; that is why the potassium flow follows the

sodium flow.

The question remains: how does the nerve membrane suddenly begin to leak

ions that it barred only a second before? Part of the answer has come from

experiments using a nerve poison called tetrodotoxin (TTX). It is

well-known in Japan as the ingredient of fugu, the puffer fish, that numbs

the taste buds or, if the chef is careless, kills. TTX blocks sodium

channels. Caesium blocks the potassium channels. If a nerve is bathed in

TTX and caesium, there should be no membrane current at all.

At the beginning of the 1970s, two groups of scientists–Clay Armstrong and

Pancho Bezanilla in America, Dr Keynes and Eduardo Rojas in

Britain–managed to measure the tiny current that does flow for a fraction

of a millisecond under these conditions. They called this the gating

current. It flows when, under the influence of a voltage across the

membrane, charged molecular plugs break away to unblock the channels.

Research today concentrates on matching what is known of the molecular

structure of the channels, with ever finer readings of their electrical

behaviour, to discover how and why the channels open and close. This

continues the escape from “biological generalisations”, in favour of Dr

Hodgkin’s and Dr Huxley’s approach.