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.