| By Nancy Fliesler
TODAY, ANIMAL ELECTRICITY
is proving to be more important than anyone ever
thought, and Children's Hospital Boston researcher David
Clapham, MD, PhD, is at the forefront of studying it.
Put simply, says Clapham, "all cells are batteries,"
and fully 30 percent of a cell's energy is spent keeping those batteries
charged. The cell stores up its energy by keeping some electrically
charged particles, or ions, inside its boundaries, and keeping other
ions out. Clapham, director of Basic Cardiac Research at Children's
and a Howard Hughes investigator, has devoted his career to studying
these cellular batteries, particularly the "switches"
that turn them on and off.
These switches, better known as ion channels, are roughly donut-shaped
proteins that straddle the membrane of every cell in the body. They're
the gatekeepers that let ions flow in and out of the cell. Any given
cell might have hundreds or thousands of channels. The right stimulus„a
messenger molecule, a change in voltage, or even a change in acidity
or temperature„can throw a channel open, allowing ions to pass through.
As in a battery, opposites attract: positively-charged ions move
toward a negative charge, and vice versa, and the surge of ions
across the cell membrane generates tiny electrical currents that
orchestrate a multitude of bodily functions.
"The working of every cell in the body requires ion channels,"
Clapham says. "They govern where cells go and how they signal
each other."
First discovered in the 1940s, ion channels have been implicated
in a long list of diseases, including cystic fibrosis, diabetes,
cardiac arrhythmias, neurologic and psychiatric diseases, gastrointestinal
disorders, and hypertension (see figure below). A malfunctioning
channel can throw off the timing of a heartbeat, cause a brain cell
to "talk" too much or too little to its neighbors, or
constrict a blood vessel too tightly. Many drugs on the market today
act on ion channels, either directly or indirectly, including Valium,
Glucotrol for diabetes, Robitussin cough medicine, and even the
hair-loss drug Rogaine.
Channels come in several types, based on the ions that tunnel through
them: positively charged sodium, calcium, or potassium ions, or
negatively charged chloride ions. Sodium channels are excitatory,
tending to trigger fast physiologic responses; potassium channels
are inhibitory, tending to slow things down. Regardless of their
effect, the signals that pass through ion channels are lightning
fast.
Using ion channels, cells continually adjust their inside and outside
electrical charges. Normal levels of calcium ions, for example,
are 20,000 times higher outside the cell than inside. Heart cells,
using special voltage-sensitive calcium channels, can quickly upset
this ratio, generating the spike of energy needed for a heartbeat.
Through the controlled movement of ions across its membrane, the
cell returns to its resting state and then recharges its battery
to begin the next cycle.
Clapham, who originally studied electrical engineering, wants to
know what triggers different kinds of channels to open and close,
how channels detect these triggers, how channels "know"
to let one kind of ion pass but not another, and how they physically
open and close.
His work began in the 1970s, as the field was undergoing dramatic
change. He did his postdoctoral studies in Germany with Erwin Neher,
who together with another researcher named Bert Sakmann, would win
the Nobel Prize for developing the patch clamp technique. Patch
clamping allows researchers to study the real-time electrical behavior
of a single cell„and sometimes even a single ion channel„by directly
measuring the current.
"Many biologists are used to looking at things that are dead,
frozen, fixed, or as pieces of the whole," Clapham says. "An
ion channel is something that's live in its environment„you can
see it working, while it's doing its job."
Scott Ramsey, a postdoctoral fellow in Clapham's lab, demonstrates
the patch clamp technique. Using a joystick, and peering through
a microscope, he carefully eases a needle-thin probe against the
membrane of a living embryonic kidney cell, forming a tight seal.
A small jagged line of current shows up on his computer screen,
amplified to make it visible. He then adds a chemical known to activate
ion channels. As the chemical diffuses in, there's a spike of current
on the screen„a few trillionths of an amp„that Ramsey measures and
records. "That's how the channels talk to us," he says.
"That's their language."
The 1980s brought a second key advance: the ability to clone ion
channels and determine the makeup of their genes. This led to an
explosion of research, as investigators tampered with ion channel
genes„or completely disabled them„to alter the channels and watch
what happened.
A third revolution came in 1998, when a team led by Rod MacKinnon,
a researcher at Rockefeller University, took an ion channel protein
(in this case a potassium channel), grew it in crystals with a lattice-like
structure, and aimed an X-ray beam through the crystals. The way
the X-rays bounced off the lattice revealed, for the first time,
the channel's three-dimensional shape. It was known that ion channels
have sensing mechanisms that pick up triggering cues, a pore through
which the ions flow, and a selectivity filter that allows only certain
types of ions through, but those structures had never been seen.
The 3-D, extremely high-resolution images from X-ray crystallography
are accurate down to the atom.
Clapham praises the work of his friend and colleague, MacKinnon,
but adds, "There's so much more to do." First on his list
is to crystallize the "NaChBac" channel, which his lab
discovered and cloned. NaChBac (short for sodium [Na] channel of
bacteria) is activated by changes in voltage. If his lab can coax
NaChBac to grow in crystals, Clapham hopes that X-ray imaging will
reveal, structurally, how the voltage sensor works. It should also
reveal the workings of the sodium selectivity filter, which admits
sodium but excludes ions like potassium that are very close in size.
The sodium filter is key to the signaling of "excitable cells"
like nerve and muscle cells, which not only use electricity internally
but can fire off electrical impulses (known as action potentials)
to their neighbors.
In Clapham's lab, and in others throughout Children's, ion channel
research is touching areas as wide-ranging as sickle cell disease,
brain development and craniofacial development (see sidebar). But
Clapham's most recent discovery is a calcium channel found only
in the tails of sperm. Dubbed CatSper, it provides the wriggle and
thrust that propel sperm toward the egg; without it, sperm are incapable
of fertilization. Hydra Biosciences, Inc., co-founded by Clapham,
is now developing a male contraceptive that would specifically target
CatSper. Since CatSper is unique to sperm, Clapham says, such a
drug shouldn't have side effects.
Some of the hottest
ion channel research involves non-excitable cells:
cells with no obvious electrical activity. Clapham is now focusing
on a family of channels known as transient receptor potential (TRP)
channels. Preliminary research indicates that many TRP channels
are involved in sensory functions like smell, taste, hearing, primitive
forms of vision and even pheromone sensing. Last year, Clapham's
lab reported that TRP channel TRPV3 is activated by subtle temperature
changes. Found in skin, hair follicles, and nerve cells, it may
help regulate body temperature. A still-mysterious group of TRP
channels seem to influence cells' ability to move and travel in
the body, potentially affecting functions as diverse as wound healing,
infection fighting, embryonic development and even the metastasis
of cancers.
Since ion channels are major drug targets, the pharmaceutical industry
has invested heavily in studying them. Clapham and collaborator
Dejian Ren, a researcher from the University of Pennsylvania, recently
filed a patent application for the use of NaChBac, the bacterial
channel, as a template for testing channel blockers and openers.
NaChBac is easy to manipulate in the lab. Through genetic engineering,
selected channel components can be removed, and their counterparts
from human (or other mammalian) channels can be inserted in their
place and tested. Alternatively, a sodium channel could be converted
into, say, a calcium channel by changing the pore and filter. Such
a tool would be convenient not just for drug discovery, but for
learning more about how channels work, Clapham says.
Yet despite the commercial interest, ion channels are surprisingly
under-appreciated by the general medical community. For a start,
people who gravitate to life science tend not to be interested in
electronics or physical science, says Clapham. And the study of
ion channels requires electrophysiology tools and techniques that
are unfamiliar to researchers more used to cloning and expressing
genes.
"Most people in biology don't like to think about ion channels
because they involve using electrodes and knowing what volts and
amps are," Clapham says. "But they are how we work."
To support Dr. Clapham's ion channel research
contact Karen Ann Engelbourg in the Children's
Hospital Trust at (617) 355-8863 or karen.engelbourg@chtrust.org
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