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SCIENCEWEEK

Neurobiology - Part 1

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Section 1

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Thematic Issue: Ion Channels

1. Introduction
2. On Sodium and Potassium Ion Channels
3. On Voltage-Gated Potassium Channels
4. Structure and Mechanism of a Calcium-Gated Potassium Channel
5. On the Open-Pore Conformation of Potassium Channels
6. On Acid-Sensitive Ion Channels
7. Ion Channel Proteins and Calcium Channels
8. Artificial Membrane Receptors and Ion Channels
9. Ion Channels, Synaptic Transmission, and Long-Term
Potentiation

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Section 2

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1. INTRODUCTION.

AXONS TO SYNAPSES.

"Even early in the 20th century, it was already understood that
nerve cells have an electrical potential, the resting membrane
potential, across their membrane, and that signaling along the
axon is conveyed by a propagated electrical signal, the action
potential, which was thought to nullify the resting potential. In
1937 Alan Hodgkin discovered that the action potential gives rise
to local current flow on its advancing edge and that this current
depolarizes the adjacent region of the axonal membrane
sufficiently to trigger a traveling wave of depolarization. In
1939 Hodgkin and Andrew Huxley made the surprising discovery that
the action potential more than nullifies the resting potential --
it reverses it. Then, in the late 1940s, Hodgkin, Huxley, and
Bernard Katz explained the resting potential and the action
potential in terms of the movement of specific ions -- potassium
(K+), sodium (Na+), and chloride (Cl-) -- through pores (ion
channels) in the axonal membrane. This ionic hypothesis unified a
large body of descriptive data and offered the first realistic
promise that the nervous system could be understood in terms of
physicochemical principles common to all of cell biology. The
next breakthrough came when Katz, Paul Fatt, and John Eccles
showed that ion channels are also fundamental to signal
transmission across the synapse. However, rather than being gated
by voltage like the Na+ and K+ channels critical for action
potentials, excitatory synaptic ion channels are gated chemically
by ligands such as the transmitter acetylcholine. During the
1960s and 1970s, neuroscientists identified many amino acids,
peptides, and other small molecules as chemical transmitters,
including acetylcholine, glutamate, GABA, glycine, serotonin,
dopamine, and norepinephrine. On the order of 100 chemical
transmitters have been discovered to date."

E.R. Kandel and L.R. Squire: Science 2000 290:1113

ION CHANNELS.

"The generation of electrical signals in neurons requires both
selective membrane permeability and specific ion concentration
gradients across the plasma membrane. The membrane proteins that
give rise to these two essential conditions are called ion
channels and pumps, respectively. Ion channels, as the phrase
implies, have pores that allow particular ions to diffuse across
the neuronal membrane. Some channels also have specialized
domains that sense the electrical potential across the membrane.
Such channels open or close in response to the level of membrane
potential, allowing the membrane permeability to be voltage-
sensitive. Many types of voltage-sensitive ion channels have been
identified, and this diversity generates a wide spectrum of
electrical characteristics among neuron types. Pumps are membrane
proteins that produce and maintain ion concentration gradients.
The Na+ pump, which regulates the intracellular concentrations of
both Na+ and K+ by hydrolyzing ATP to fuel the translocation of
these ions across the plasma membrane, is the best-studied
example. Other pumps produce concentration gradients for a
variety of other ions. From the perspective of neural signaling,
pumps and channels are complementary: pumps create the
concentration gradients that impel ions to diffuse through open
channels, thus generating electrical signals."

D. Purves et al (eds.): Neuroscience 1997 Sinauer p. 69.

CHANNELS VS. TRANSPORTERS

"Physiologists say that ions and neutral solutes can cross
biological membranes via "transporters" and "channels". We tend
to think about the difference between transporters and channels
in terms of gating mechanisms. Ion channels exhibit a wide range
of selectivity properties and permeation rates, but their gating
at the most basic level can be thought of in terms of a single
barrier or gate acting as a switch. When the gate is closed, ions
can't permeate; when the gate is opened, a permeation pathway for
ions allows flux, often at very high rates (up to 10^(8)/sec).
Transporters and ion pumps, on the other hand, mediate flux that
can be explained better by the presence of two gates -- one
external and one internal. In this canonical transport scheme,
the two gates are never open simultaneously. Instead they open
sequentially to allow the cytoplasmic and extracellular
compartments alternating access to the permeation pathway. Unlike
flux through an open ion channel, there must be a gating cycle
every time solute is transported, so transporters generally
mediate much slower rates of solute permeation (sometimes as slow
as 1/sec). An alternating access model can explain how a
neurotransmitter can be accumulated against its electrochemical
gradient if other ions are stoichiometrically co- or counter-
transported down their gradients. The kinetic scheme is formally
equivalent to that of a carrier (like valinomycin) that shuttles
back and forth across the membrane, although the physical process
is quite distinct."

Michael P. Kavanaugh: Proc. Nat. Acad. Sci. 1998 95:12737.

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2. SODIUM AND POTASSIUM ION CHANNELS.

STRUCTURE OF THE VOLTAGE-GATED SODIUM CHANNEL.

Electrical signals in the nervous system are generated by the
movement of ions across the nerve cell membrane. These ionic
currents flow through aqueous pores of membrane proteins known as
"ion channels", and these channels vary in ion selectivity, with
some channels specifically permeable to sodium or potassium or
calcium ions.     Certain sodium, potassium, and calcium channels
are "voltage-gated" -- their permeability is switched on and off
by changes in the potential difference across the cell membrane -
-and these channels essentially control the dynamic electric
activity of nerve and muscle cells.     The voltage-activated
sodium channel from eel electric organs (of relevance in this
report) is a single molecule of approximately 1800 amino acids,
within which are 4 repeating domains. These domains are
architecturally equivalent to the subunits of other ion channels.
Within each domain, there are apparently 6 membrane spanning
regions connected by intracellular and extracellular polypeptide
loops. The eel channel is apparently representative of a diverse
family of channel proteins present in nerve and muscle fibers.   
Fifty years ago, the existence of such specific protein channels
was not even conceived, although it was recognized there had to
be some explanation for specific ion currents in nerve and muscle
cells. Thirty years ago, the existence of such channels was
vaguely proposed, but with hardly any molecular information. In
the past two decades, ion channels have become definitive
structural entities.

C. Sato et al (7 authors at 5 installations, JP CH) present a
report on the structure of the sodium-sensitive ion channel, the
authors making the following points:

1) The authors point out that the voltage-sensitive sodium,
potassium, and calcium channels operate together to amplify,
transmit, and generate electric pulses in animals. Sodium and
calcium channels are involved in cell excitation, neuronal
transmission, muscle contraction, and many functions that relate
directly to human diseases. Sodium channels, which are
glycosylated proteins with a relative molecular mass of
approximately 300,000, are responsible for signal transduction
and amplification, and are chief targets of anesthetic drugs and
neurotoxins.

2) The authors report the 3-dimensional structure of the voltage-
sensitive sodium channel of the eel  Electrophorus electricus.
The structure was determined at 19 angstroms resolution by
helium-cooled cryo-electron microscopy and single-particle image
analysis of the solubilized sodium channel. The authors report
the channel has a bell-shaped outer surface of 135 angstroms in
height and 100 angstroms in side length at the square-shaped
bottom, and a spherical top with a diameter of 65 angstroms.
Several inner cavities are connected to 4 small holes and 8
orifices close to the extracellular and cytoplasmic membrane
surfaces. Homologous voltage-sensitive calcium and tetrameric
potassium channels, which regulate secretory processes and the
membrane potential, may possess related structure.

Nature 2001 409:1047

Related Background:

NEUROBIOLOGY: ATOMIC SCALE MOVEMENTS IN POTASSIUM CHANNELS.

The functional electrical activity of nerve cells is based
essentially on the rapid movements of ions across the membranes
of these cells, especially the movements of sodium and potassium
ions. These ion movements occur through special pores ("ion
channels") in the cell membrane, and one of the important
problems during the past few decades has been to characterize
these ion channels at the molecular level. Most ion channels are
selective, allowing only ions of a certain type to pass, and an
individual cell has ion channels with various ion selectivities.
The selectivity of an ion channel can be "gated", the channel
effectively opened or closed, and ion channels are said to
"voltage-gated" or "ligand-gated", depending on how the change in
selectivity is provoked. The term "voltage-gated" refers to the
opening or closing of an ion channel by changes in the electrical
potential across the membrane, while the term "ligand-gated"
refers to opening and closing of an ion channel by interactions
between ligands and membrane receptors. It has become apparent
that voltage-gated ion channels are transmembrane proteins
consisting of 4 identical subunits, each of which contains 6
transmembrane segments. Studies of the potassium ion channel have
identified two segments that contain several charged protein
residues, and these charged residues apparently sense changes in
the potential difference across the membrane and form part of the
membrane "voltage sensor". Although these regions apparently
undergo conformational changes in response to changes in membrane
potential, little is known about the nature of these changes.

A. Cha et al (4 authors at 2 installations, US) report a study of
molecular movements of the voltage-sensing region in a potassium
channel, the authors making the following points:

1) The authors used *lanthanide-based fluorescence resonance
energy transfer to measure distances between *Shaker potassium-
channel protein subunits at specific residues. Voltage dependent
distance changes of up to 3.2 angstroms were measured at several
sites near one of the charged protein segments (S4). These
movements directly correlated with electrical measurements of the
voltage sensor, establishing a link between physical changes and
electrical charge movement.

2) The authors suggest that the measured distance changes
indicate that the region associated with the S4 segment undergoes
a rotation and possible tilt, rather than a large transmembrane
movement, in response to voltage.

3) The authors conclude: "These results demonstrate the first in
situ measurement of atomic scale movement in a transmembrane
protein."

In a contiguous and related report, K.S. Glauner et al (4 authors
at University of California Berkeley, US) present a study of
voltage-sensor movements of a potassium channel, the authors
making the following points:

1) The authors used fluorescence resonance energy transfer as a
"spectroscopic ruler" to determine distances between S4 subunits
in the Shaker potassium channel in different gating states.

2) The authors conclude their experimental evidence is consistent
with the S4 subunit being a tilted helix that twists during
activation. The authors propose that helical twist contributes to
the movement of charged side chains across the membrane electric
field and that this movement is involved in coupling voltage-
sensing to gating.

Nature 1999 402:809,813

(Nature 16 Dec 99 402:813)

Text Notes:

... ... *lanthanide-based fluorescence resonance energy transfer:
The term "fluorescence resonance energy transfer" (also called
"fluorescence energy transfer") refers to energy transfer between
two fluorophores (chemical groups or molecules capable of
fluorescence). If the two fluorophores are attached to a molecule
at different positions, observations of fluorescence energy
transfer between them can be used to determine the distance
between the two attachment positions. Lanthanide-based resonance
energy transfer is a modification of conventional fluorescence
resonance energy transfer in which a long-lived lanthanide donor
transfers energy in a distance-dependent manner to a conventional
organic fluorescent acceptor. This technique has previously been
used to measure angstrom-scale conformational changes in
proteins.

... ... *Shaker potassium-channel protein: The term "Shaker" here
refers to a mutant of the fruit fly Drosophila, the mutant
exhibiting intense shaking of the legs and body in response to
exposure to a volatile anesthetic. Genetic analysis of the
mutation some years ago led to the sequencing of a Drosophila
gene expressing a potassium-channel protein, the shaking of the
insect apparently resulting from a mutation in this gene, with
the mutation producing long-lasting potassium ion currents when
nerve fibers are activated. Once the Shaker gene was sequenced, a
conventional procedure was developed in the 1980s to have this
gene expressed in frog egg cells (oocytes) in order to advance
the study of the behavior of potassium ion channels. Thus, the
potassium ion channels in this report are ion channels derived
from the fruit fly Drosophila and expressed in the frog Xenopus
laevis egg cell. The essential aspects of frog oocytes is that
they are large cells (up to 1 millimeter in diameter), and the
introduction of foreign *messenger RNA into the egg cell readily
results in the production of the protein encoded by the
introduced messenger RNA.

 ... ... *messenger RNA: (mRNA) The ribonucleic acid molecule
transcribed from DNA that carries the coded information
specifying the sequence of amino acids in a protein.

Related Background:

STRUCTURAL REARRANGEMENTS IN POTASSIUM CHANNEL GATING.

Ion channels are protein channels in cell membranes that allow
ions to pass from extracellular solution to intracellular
solution and vice versa. Most ion channels are selective,
allowing only certain ions to pass, and an individual cell has
ion channels with various ion selectivities. The selectivity of
an ion channel can be "gated", the channel effectively opened or
closed, and ion channels are said to voltage-gated or ligand-
gated, depending on how the change in selectivity is provoked.
The opening of a previously closed ion channel produces a sudden
increase in transmembrane conductance for that ion, and the
process is called "activation". The gating of the movements of
ions through ion channels is of considerable importance for
various processes in all living systems, and forms the basis of
the electrical activity of all nervous systems. Recently (see
background material below), an important advance in ion-channel
research occurred with the experimental determination of the
crystal structure of a potassium channel (KcsA) in the bacterial
species  Streptomyces lividans. The structure involves a
tetrameric complex with a centrally located pore framed by the
apposition of individual subunits, each subunit with 2
transmembrane helices (TM1 and TM2) flanking a "selectivity
filter". Intensive studies of this potassium channel in *planar
lipid bilayers have been in progress in a number of laboratories.

E. Porozo et al (3 authors at University of Virginia, U) report a
study of the structural rearrangements underlying activation
gating in this potassium channel, the study using *spin-labeling
methods and *electron paramagnetic resonance spectroscopy. The
authors report that a comparison of the closed and open
conformations of the channel revealed periodic changes in spin-
label mobility and intersubunit *spin-spin interaction consistent
with rigid-body movements of the two transmembrane helices TM1
and TM2. These changes involve translations and counterclockwise
rotations of both helices relative to the center of symmetry of
the channel. The movement of TM2 apparently increases the
diameter of the permeation pathway along the point of convergence
of the four subunits, thus opening the pore. Although the
extracellular residues flanking the selectivity filter remained
immobile during gating, small movements were detected at the *C-
terminal end of the pore helix, and the authors suggest this has
possible implications for the gating mechanism.

Science 1999 285:73

Text Notes:

... ... *planar lipid bilayers: The cell membrane consists of a
lipid bilayer and associated proteins, the ensemble approximately
75 to 100 angstroms in thickness. Similar membranes are also
found within a cell surrounding various organelles. Lipid
bilayers are spontaneously forming self-organizing bimolecular
layers of certain molecules (lipids) with long nonpolar chains
terminated by a polar group. In addition to their presence in
cell membranes, such molecules (surfactants) are also found in
soaps. A variety of artificial lipid bilayer membrane systems can
be investigated in the laboratory.

... ... *spin-labeling methods: A "spin-label" is a synthetic
paramagnetic organic free radical incorporated in a macromolecule
or assemblage of macromolecules and used, in particular, in
electron paramagnetic resonance spectroscopy.

... ... *electron paramagnetic resonance spectroscopy: (ESR) This
technique is used to investigate paramagnetic centers in a
molecular system. Only electrons whose spin is not paired with
the oppositely directed spin of another electron give an ESR
signal. With this technique, information can be obtained about
certain transitional ions, free radicals, and free electron
centers. A probe giving an ESR signal can be incorporated into
membrane lipids or attached to proteins to enable otherwise
inaccessible systems to be studied. Through analysis of ESR
spectra, rates of molecular motion and relative orientation of
spin-labeled molecules whose motion is restrained by surrounding
molecules can be determined. Measurements of rates of molecular
motion and molecular orientation have proved to be important in
the study of a variety of biological problems.

... ... *spin-spin interaction: In this context, an interaction
of two neighboring paramagnetic entities, the interaction
producing a change in ESR signal.

... ... *C-terminal end: In general, this refers to the end of
any polypeptide chain at which the 1-carboxy function of a
constituent alpha-amino acid is not attached in peptide linkage
to another amino acid residue.

Related Background Brief:

ANALYSIS OF POTASSIUM ION MEMBRANE CHANNEL STRUCTURE. The
potassium ion channel from the prokaryotic soil bacterium
Streptomyces lividans is an integral membrane protein with
sequence similarity to all known potassium ion channels,
particularly in the pore region. Doyle et al (8 authors at
Rockefeller University, US) report an x-ray analysis (data to 3.2
angstroms) of the Streptomyces lividans potassium channel reveals
four identical subunits create an inverted cone cradling the
selectivity filter of the pore in its outer end. The narrow
selectivity filter is only 12 angstroms long, whereas the
remainder of the pore is wider and lined with hydrophobic amino
acids. The selectivity filter is apparently held open by
structural constraints to coordinate potassium ions but not
smaller sodium ions. The authors suggest the architecture of the
pore establishes the physical principles underlying selective
potassium ion conduction. Science 1998 280:69

Related Background Brief:

SIMILAR STRUCTURE OF PROKARYOTIC VS. EUKARYOTIC K(+) CHANNELS.
Toxins from scorpion venom are known to interact with potassium
ion channels in eukaryotic cell membranes. Mackinnon et al (5
authors at Rockefeller University, US) report the use of resin-
attached mutant potassium ion channels from the bacterium
Streptomyces lividans to screen scorpion venom, and the toxins
that interact with the channel were identified by mass
spectrometry. The authors suggest their results indicate that the
prokaryotic potassium ion channel, whose structure has now been
revealed, has the same pore structure as eukaryotic potassium ion
channels, and that this structural conservation, through the
application of their techniques, offers a new approach to
potassium ion channel pharmacology. Science 1998 280:106

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3. ON VOLTAGE-GATED POTASSIUM CHANNELS

Gary Yellen (Harvard University, US) discusses voltage-gated
potassium channels, the author making the following points:

1) The voltage-gated K+ channels are the prototypical voltage-
gated channels. At their simplest, they are homotetrameric
channels, with each subunit containing a voltage sensor and
contributing to the central pore. The standard K+ channel subunit
contains six transmembrane regions, with both amino and carboxy
termini on the intracellular side of the membrane (a tetrameric
"6TM architecture"). The pore-forming subunits of voltage-gated
Na+ and Ca2+ channels contain four non-identical repeats of this
motif, strung together on a single polypeptide. There is enormous
variety within each of these channel families: voltage-gated K+
channels alone are made by at least 22 different genes in
mammals, with additional variety produced by alternative splicing
and heteromultimerization.

2) Some close relatives of the voltage-gated K+ channels share
the tetrameric 6TM architecture but differ in key functional
features. These include the sensory channels of the
photoreceptors and olfactory neurons, which are non-selective
(Na+ and K+) channels that are cyclic-nucleotide-gated (the "CNG
channels") and insensitive to voltage, and the pacemaker channels
found in heart muscle and neurons (the "HCN channels") that are
controlled by both cyclic nucleotides and voltage.

3) Each of these signaling proteins performs three main
functions. Ion permeation is the central function, and the
movement of ions through the pore must be fast and ion-selective.
This permeation is then regulated by opening and closing of the
pore -- a set of conformational changes called "gating". Finally,
the gating is coupled to a sensing mechanism, which detects
transmembrane voltage in the core members of the family, but can
also be geared to sense Ca2+, cyclic nucleotides, and perhaps
other cellular signals. From approximately 40 years of
electrophysiology and biophysics, a little over a decade of
cloning followed by mutagenesis and physiology on cloned
channels, and from a few important crystal structures of some
bacterial relatives of these channels, we now have a relatively
clear picture of how these functions are accomplished.(1-5)

References (abridged):

1. Hodgkin, A. L. & Huxley, A. F. A quantitative description of
membrane current and its application to conduction and excitation
in nerve. J. Physiol. (Lond.) 117, 500-544 (1952)

2. Latorre, R. & Miller, C. Conduction and selectivity in
potassium channels. J. Membr. Biol. 71, 11-30 (1983)

3. Doyle, D. A. et al. The structure of the potassium channel:
molecular basis of potassium conduction and selectivity. Science
280, 69-77 (1998)

4. Zhou, Y., Morais Cabral, J. H., Kaufman, A. & MacKinnon, R.
Chemistry of ion hydration and coordination revealed by a K+
channel-Fab complex at 2.0 � resolution. Nature 414, 43-48 (2001)

5. Morais-Cabral, J. H., Zhou, Y. & MacKinnon, R. Energetic
optimization of ion conduction rate by the K+ selectivity filter.
Nature 414, 37-42 (2001)

Nature 2002 419:35

Related Background Brief:

THE STRUCTURE OF THE POTASSIUM CHANNEL: MOLECULAR BASIS OF K+
CONDUCTION AND SELECTIVITY. The potassium channel from
Streptomyces lividans is an integral membrane protein with
sequence similarity to all known K+ channels, particularly in the
pore region. The authors report that x-ray analysis with data to
3.2 angstroms reveals that four identical subunits create an
inverted teepee, or cone, cradling the selectivity filter of the
pore in its outer end. The narrow selectivity filter is only 12
angstroms long, whereas the remainder of the pore is wider and
lined with hydrophobic amino acids. A large water-filled cavity
and helix dipoles are positioned so as to overcome electrostatic
destabilization of an ion in the pore at the center of the
bilayer. Main chain carbonyl oxygen atoms from the K+ channel
signature sequence line the selectivity filter, which is held
open by structural constraints to coordinate K+ ions but not
smaller Na+ ions. The selectivity filter contains two K+ ions
about 7.5 angstroms apart. This configuration promotes ion
conduction by exploiting electrostatic repulsive forces to
overcome attractive forces between K+ ions and the selectivity
filter. The architecture of the pore establishes the physical
principles underlying selective K+ conduction. D.A. Doyle et al:
Science 1998 280:69.

Related Background Brief:

CHEMISTRY OF ION COORDINATION AND HYDRATION REVEALED BY A K+
CHANNEL-FAB COMPLEX AT 2.0 A RESOLUTION. Ion transport proteins
must remove an ion's hydration shell to coordinate the ion
selectively on the basis of its size and charge. To discover how
the K+ channel solves this fundamental aspect of ion conduction,
the authors report they solved the structure of the KcsA K+
channel in complex with a monoclonal Fab antibody fragment at 2.0
A resolution. The authors demonstrate how the K+ channel
displaces water molecules around an ion at its extracellular
entryway, and how it holds a K+ ion in a square antiprism of
water molecules in a cavity near its intracellular entryway.
Carbonyl oxygen atoms within the selectivity filter form a very
similar square antiprism around each K+ binding site, as if to
mimic the waters of hydration. The selectivity filter changes its
ion coordination structure in low K+ solutions. This structural
change is crucial to the operation of the selectivity filter in
the cellular context, where the K+ ion concentration near the
selectivity filter varies in response to channel gating. Y. Zhou
et al: Nature 2001 414:43.

Related Background Brief:

THE VOLTAGE SENSOR IN VOLTAGE-DEPENDENT ION CHANNELS. In voltage-
dependent Na, K, or Ca channels, the probability of opening is
modified by the membrane potential. This is achieved through a
voltage sensor that detects the voltage and transfers its energy
to the pore to control its gate. The author presents the
theoretical basis of the energy coupling between the electric
field and the voltage, which allows the interpretation of the
gating charge that moves in one channel. Movement of the gating
charge constitutes the gating current. The properties are
described, along with macroscopic data and gating current noise
analysis, in relation to the operation of the voltage sensor and
the opening of the channel. Structural details of the voltage
sensor operation were resolved initially by locating the residues
that make up the voltage sensor using mutagenesis experiments and
determining the number of charges per channel. The changes in
conformation are then analyzed based on the differential exposure
of cysteine or histidine-substituted residues. Site-directed
fluorescence labeling is then analyzed as another powerful
indicator of conformational changes that allows time and voltage
correlation of local changes seen by the fluorophores with the
global change seen by the electrophysiology of gating currents
and ionic currents. Finally, the author describes the novel
results on lanthanide-based resonance energy transfer that show
small distance changes between residues in the channel molecule.
All of the electrophysiological and the structural information
are finally summarized in a physical model of a voltage-dependent
channel in which a change in membrane potential causes rotation
of the S4 segment that changes the exposure of the basic residues
from an internally connected aqueous crevice at hyperpolarized
potentials to an externally connected aqueous crevice at
depolarized potentials. F. Bezanilla: Physiol Rev 2000
Apr;80(2):555.

Related Background Brief:

STRUCTURE AND FUNCTION OF CYCLIC NUCLEOTIDE-GATED CHANNELS.
Cyclic nucleotide-gated (CNG) channels play important roles in
both visual (Yau & Baylor 1989) and olfactory (Zufall et al 1994)
signal transduction. The cloning of the gene coding for a rod
photoreceptor channel (Kaupp et al 1989) and the subsequent
cloning of related genes from olfactory neurons (Dhallan et al
1990, Ludwig et al 1990, Goulding et al 1992) has sparked much
progress over the past several years in elucidating the
structural bases for the function of the CNG channels. One of the
surprising features to emerge from these cloning studies was that
the CNG channels are structurally homologous to the voltage-gated
channels (Jan & Jan 1990) despite the fact that the CNG channels
are gated by the binding of a ligand-cAMP or cGMP-and not by
voltage. The authors focus on recent studies of the relationship
between the structure and function of the CNG channels. Given the
homology between CNG channels and voltage-gated channels, such
studies are likely to provide important general information about
the structure and function of a wide variety of channel types.
W.N. Zagotta and S.A. Siegelbaum: Annu Rev Neurosci 1996 19:235.

Related Background Brief:

MOLECULAR DYNAMICS OF KCSA IN A PHOSPHOLIPID BILAYER. Potassium
channels enable K(+) ions to move passively across biological
membranes. Multiple nanosecond-duration molecular dynamics
simulations (total simulation time 5 ns) of a bacterial potassium
channel (KcsA) embedded in a phospholipid bilayer reveal motions
of ions, water, and protein. Comparison of simulations with and
without K(+) ions indicate that the absence of ions destabilizes
the structure of the selectivity filter. Within the selectivity
filter, K(+) ions interact with the backbone (carbonyl) oxygens,
and with the side-chain oxygen of T75. Concerted single-file
motions of water molecules and K(+) ions within the selectivity
filter of the channel occur on a 100-ps time scale. In a
simulation with three K(+) ions (initially two in the filter and
one in the cavity), the ion within the central cavity leaves the
channel via its intracellular mouth after approximately 900 ps;
within the cavity this ion interacts with the O-gamma atoms of
two T107 side chains, revealing a favorable site within the
otherwise hydrophobically lined cavity. Exit of this ion from the
channel is enabled by a transient increase in the diameter of the
intracellular mouth. Such "breathing" motions may form the
molecular basis of channel gating. I.H. Shrivastava and M.S.
Sansom: Biophys J 2000 78:557.

Related Background Brief:

ENERGETICS OF ION CONDUCTION THROUGH THE K+ CHANNEL. K+ channels
are transmembrane proteins that are essential for the
transmission of nerve impulses. The ability of these proteins to
conduct K+ ions at levels near the limit of diffusion is
traditionally described in terms of concerted mechanisms in which
ion-channel attraction and ion-ion repulsion have compensating
effects, as several ions are moving simultaneously in single file
through the narrow pore. The efficiency of such a mechanism,
however, relies on a delicate energy balance -- the strong ion-
channel attraction must be perfectly counterbalanced by the
electrostatic ion-ion repulsion. To elucidate the mechanism of
ion conduction at the atomic level, the authors performed
molecular dynamics free energy simulations on the basis of the X-
ray structure of the KcsA K+ channel. The authors report they
find that ion conduction involves transitions between two main
states, with two and three K+ ions occupying the selectivity
filter, respectively; this process is reminiscent of the "knock-
on" mechanism proposed by Hodgkin and Keynes in 1955. The largest
free energy barrier is on the order of 2-3 kcal per mol, implying
that the process of ion conduction is limited by diffusion. Ion-
ion repulsion, although essential for rapid conduction, is shown
to act only at very short distances. The calculations show also
that the rapidly conducting pore is selective. S. Berneche and
B.Roux: Nature 2001 414:23.

Related Background Brief:

POTASSIUM AND SODIUM BINDING TO THE OUTER MOUTH OF THE K+
CHANNEL. Molecular dynamics simulations of the K+ channel from
Streptomyces lividans (KcsA channel) were performed in a
membrane-mimetic environment with Na+ and K+ in different initial
locations. The structure of the channel remained stable and well
preserved for simulations lasting up to 1.5 ns. Salt bridges
between Asp80 and Arg89 of neighboring subunits, not detected in
the X-ray structure, enhanced the stability of the tetrameric
structure. Na+ or K+ ions located in the channel vestibule lost
part of their hydration shell and diffused into the channel inner
pore in less than a few hundred picoseconds. This powerful
catalytic action was caused by strong electrostatic interactions
with Asp80 and Glu71. The hydration state of the metal ions
turned out to depend significantly on the conformational
flexibility of the channel. Furthermore, Na+ entered the channel
inner pore bound to more water molecules than K+. The different
hydration state of the two ions may be a determinant factor in
the ion selectivity of the channel. L. Guidoni et al:
Biochemistry 1999 38:8599.

Related Background Brief:

THE CAVITY AND PORE HELICES IN THE KCSA K+ CHANNEL: ELECTROSTATIC
STABILIZATION OF MONOVALENT CATIONS. The electrostatic influence
of the central cavity and pore alpha helices in the potassium ion
channel from Streptomyces lividans (KcsA K+ channel) was analyzed
by solving the finite difference Poisson equation. The cavity and
helices overcome the destabilizing influence of the membrane and
stabilize a cation at the membrane center. The electrostatic
effect of the pore helices is large compared to that described
for water-soluble proteins because of the low dielectric membrane
environment. The combined contributions of the ion self-energy
and the helix electrostatic field give rise to selectivity for
monovalent cations in the water-filled cavity. Thus, the K+
channel uses simple electrostatic principles to solve the
fundamental problem of ion destabilization by the cell membrane
lipid bilayer. B. Roux and R. MacKinnon: Science 1999 285:100.

Related Background Brief:

LIPIDS IN THE STRUCTURE, FOLDING, AND FUNCTION OF THE KCSA K+
CHANNEL. Lipid molecules surround an ion channel in its native
environment of cellular membranes, but the importance of the
lipid bilayer and the role of lipid protein interactions in ion
channel structure and function are not well understood. The
authors report a demonstration that the bacterial potassium
channel KcsA binds a negatively charged lipid molecule. The
authors have defined the potential binding site of the lipid
molecule on KcsA by X-ray crystallographic analysis of a complex
of KcsA with a monoclonal antibody Fab fragment. The authors also
demonstrate that lipids are required for the in vitro refolding
of the KcsA tetramer from the unfolded monomeric state. The
correct refolding of the KcsA tetramer requires lipids, but it is
not dependent on negatively charged lipids as refolding takes
place in the absence of such lipids. The authors confirm that the
presence of negatively charged lipids is required for ion
conduction through the KcsA potassium channel, suggesting that
the lipid bound to KcsA is important for ion channel function.
Biochemistry 2002 41:10771.

Related Background:

ON THE POTASSIUM ION CHANNEL

M.S. Sansom and I.H. Shrivastava (University of Oxford, UK)
discuss the potassium ion channel, the authors making the
following points:

1) When the crystal structure of the bacterial channel protein
KcsA was first solved in 1998(1) , it provided us with our first
view of an ion channel at atomic resolution. The structure,
determined at the relatively modest resolution of 3.2 angstroms,
revealed the basic architecture of the channel: an extracellular
selectivity filter, a central cavity and an intracellular gate.
The importance of KcsA lies in the conservation of this basic
pore structure between KcsA and other classes of K+ channels
found in a wide range of organisms, including humans(2) . If we
can understand ion permeation in KcsA, we will thus have grasped
the fundamental mechanism of all K+ channels. But, despite a
large body of work stimulated by the earlier KcsA structure,
fundamental permeation studies were hampered by its limited
resolution.

2) New studies have extended the resolution of the KcsA structure
to 2.0 angstroms, making ions and water molecules in the filter
clearly visible(3) . Changes in structure in the presence of a
low concentration of K+ ions, and in the pattern of ion occupancy
when Rb+ ions are substituted for K+(4) , have also been
explored. These results have been combined with
electrophysiological studies of the flux of K+ versus Rb+ ions
through KcsA as a function of ionic concentration(4) . And
detailed calculations have been made of the energetics of ion
permeation through the KcsA filter(5) . Together, these new
results provide a detailed and convincing picture of the
mechanism of high throughput K+ flux   about one ion every 10
nanoseconds   through K channels. Significantly, these higher-
resolution structural studies confirm a number of predictions
from earlier simulation studies, indicating that simulations can
indeed provide new information.

3) Attempts to understand the high-throughput permeation
mechanism of KcsA have combined electrophysiological measurements
on channels reconstituted in artificial lipid bilayers with x-ray
studies of KcsA crystals in the presence of different
concentrations of K+ or Rb+ ions. Fine details of ions and water
molecules in the filter have now been revealed by the high
resolution structure of KcsA(3).

References (abridged):

1. Doyle D.A., Cabral J.M., Pfuetzner R.A., Kuo A., Gulbis J.M.,
Cohen S.L., Cahit B.T. and MacKinnon R. (1998) The structure of
the potassium channel: molecular basis of K+ conduction and
selectivity. Science, 280:69-77.

2. Lu Z., Klem A.M. and Ramu Y. (2001) Ion conduction pore is
conserved among potassium channels. Nature, 413:809-813.

3. Zhou Y., Morais-Cabral J.H., Kaufman A. and MacKinnon R.
(2001) Chemistry of ion coordination and hydration revealed by a
K+ channel-Fab complex at 2.0 � resolution. Nature, 414:43-48.

4. Morais-Cabral J.H., Zhou Y. and MacKinnon R. (2001) Energetic
optimization of ion conduction by the K+ selectivity filter.
Nature, 414:37-42.

5. BernŠche S. and Roux B. (2001) Mechanism of ions permeation
in the KcsA potassium channel. Biophys. J., 80:175a.

Current Biology 2002 12:R65

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4. STRUCTURE AND MECHANISM OF A CALCIUM-GATED POTASSIUM CHANNEL.

Y. Jiang et al (Rockefeller University, US) discuss calcium-gated
potassium channels, the authors making the following points:

1) Ion channels are central to a wide range of biological
processes including cell volume regulation, cell movement, and
electrical signal generation(1). Ion channel proteins span the
membrane of a cell, forming a conduction pathway, or pore,
through which ions diffuse down their electrochemical gradient
across the membrane. To understand how an ion channel operates as
a molecular machine, the authors addressed two mechanistic
issues. First, how do ions flow selectively through the pore, and
second, how does the pore gate, or open, in response to the
appropriate stimulus? For K+ ion channels, significant progress
has been made towards understanding the mechanism of selective
ion conduction(2-4). The authors examine the mechanism of opening
in a ligand-gated K+ channel.

2) K+ channels belong to a family of ion channels called
"tetrameric cation channels". The family includes K+, Na+, Ca2+,
cyclic nucleotide-gated, and several other ion channels. They
contain four membrane-spanning subunits or domains surrounding a
central pore that is selective for cations of one kind or
another. On the basis of the KcsA K+ channel structure(2,4), it
seems that cation selectivity is an intrinsic property of the
pore architecture, which provides a special arrangement of
cation-attractive "pore" alpha-helices probably shared by all
tetrameric cation channel family members.

3) Gating in the tetrameric cation channels is conferred through
the attachment of gating domains to the pore. In channels whose
gate opens in response to the membrane voltage (voltage-dependent
channels), an integral membrane "voltage sensor" domain is
present on each subunit(5). In ligand-gated channels, ligand-
binding domains are attached to the pore in the aqueous solution
near the membrane surface. The basic function of these gating
domains is to perform mechanical work on the ion conduction pore
to change its conformation between closed and opened states.
Thus, a voltage sensor converts energy stored in the membrane
electric field into mechanical work, whereas ligand-binding
domains convert the free energy of ligand binding into mechanical
work. Thus, ion channel gating reduces to electromechanical or
chemomechanical coupling between a gating unit and the pore unit.

4) In summary: Ion channels exhibit two essential biophysical
properties: a) selective ion conduction, and b) the ability to
gate-open in response to an appropriate stimulus. Two general
categories of ion channel gating are defined by the initiating
stimulus: ligand binding (neurotransmitter- or second-messenger-
gated channels) or membrane voltage (voltage-gated channels). The
authors present the structural basis of ligand gating in a K+
channel that opens in response to intracellular Ca2+. The authors
report they have cloned, expressed, and analysed electrical
properties, and determined the crystal structure of a K+ channel
(MthK) from Methanobacterium thermoautotrophicum in the (Ca2+)-
bound, opened state. Eight RCK domains (regulators of K+
conductance) form a gating ring at the intracellular membrane
surface. The gating ring uses the free energy of Ca2+ binding in
a simple manner to perform mechanical work to open the pore.

References (abridged):

1. Hille, B. Ion Channels of Excitable Membranes (Sinauer,
Sunderland, Massachusetts, 2001)

2. Zhou, Y., Morais-Cabral, J. H., Kaufman, A. & MacKinnon, R.
Chemistry of ion coordination and hydration revealed by a K+
channel-Fab complex at 2.0 � resolution. Nature 414, 43-48 (2001)

3. Morais-Cabral, J. H., Zhou, Y. & MacKinnon, R. Energetic
optimization of ion conduction rate by the K+ selectivity filter.
Nature 414, 37-42 (2001)

4. Doyle, D. A. et al. The structure of the potassium channel:
molecular basis of K+ conduction and selectivity. Science 280,
69-77 (1998)

5. Sigworth, F. J. Voltage gating of ion channels. Q. Rev.
Biophys. 27, 1-40 (1994)

Nature 2002 417:515

Related Background Brief:

MECHANISM OF CALCIUM GATING IN SMALL-CONDUCTANCE CALCIUM-
ACTIVATED POTASSIUM CHANNELS. The slow after-hyperpolarization
that follows an action potential is generated by the activation
of small-conductance calcium-activated potassium channels (SK
channels). The slow after-hyperpolarization limits the firing
frequency of repetitive action potentials (spike-frequency
adaptation) and is essential for normal neurotransmission. SK
channels are voltage-independent and activated by submicromolar
concentrations of intracellular calcium. They are high-affinity
calcium sensors that transduce fluctuations in intracellular
calcium concentrations into changes in membrane potential. The
authors report a study of the mechanism of calcium gating. They
find that SK channels are not gated by calcium binding directly
to the channel subunits. Instead, the functional SK channels are
heteromeric complexes with calmodulin, which is constitutively
associated with the subunits in a calcium-independent manner. The
authors suggest their data support a model in which calcium
gating of SK channels is mediated by binding of calcium to
calmodulin and subsequent conformational alterations in the
channel protein. X-M. Xia et al: Nature 1998 395:503.

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5. ON THE OPEN-PORE CONFORMATION OF POTASSIUM CHANNELS.

Four distinct subfamilies of potassium channel proteins have been
cloned from Drosophila: Shaker, Shab, Shaw, and Shal -- all with
mammalian counterparts. Each subfamily comprises various isoforms
(e.g., Shaker1, Shaker2, etc.). Isoforms from the same subfamily,
when expressed in host cells, combine to form heteromultimeric
channels, but those from different subfamilies do not.

Y. Jiang et al (Rockefeller University, US) discuss potassium
channels, the authors making the following points:

1) Potassium and other ion channels are allosteric proteins that
switch between closed and opened conformations in response to an
external stimulus in a process known as "gating". Depending on
the channel type, the gating stimulus can be the binding of a
ligand, the membrane electric field, or both. A central issue in
ion channel biophysics concerns the nature of the pore
conformational changes that accompany channel gating. What do the
opened and closed structures of the pore look like? In general,
little is known about protein conformational changes in membrane
proteins, and yet for ion channels these changes are crucial to
every aspect of their function: ion conduction, gating and
pharmacology.

2) The authors address the following questions regarding three
areas of ion channel function. a) For the conduction mechanism of
K+ channels, when the pore opens, how wide does it become, how
does opening change the electric field across the pore, and how
accessible is the K+ selectivity filter to the intracellular
solution? b) For the gating mechanism, what are the mechanics of
pore opening, are the conformational changes within the membrane
large or small? c) Finally, for K+ channel pharmacology, which
protein surfaces become exposed when the pore opens, and how
might pharmacological agents interact with the closed versus
opened state of the channel?

3) Mutational experiments from numerous laboratories have placed
important structural constraints on K+ channel gating. In
particular, mutational studies of the Shaker voltage-dependent K+
channel(1,2) -- interpreted in the context of the KcsA K+ channel
structure(3) -- identified what is probably the pore's gate. In
the KcsA structure four alpha-helices (inner helices) line the
pore's intracellular half, forming a right-handed bundle (inner
helix bundle) near the intracellular opening(3,4). The inner
helix bundle marks the point at which the Shaker pore becomes
inaccessible to thiol-reactive compounds and metal ions applied
to the cytoplasmic side of the channel when the channel is
closed1(1,5). Thus, it would seem that the extracellular half of
the pore, where the selectivity filter is located, is dedicated
to the function of K+ selectivity, and that the intracellular
half, where the pore is lined by inner helices, is dedicated to
gating.

4) In summary: Living cells regulate the activity of their ion
channels through a process known as "gating". To open the pore,
protein conformational changes must occur within a channel's
membrane-spanning ion pathway. KcsA and MthK, closed and opened
K+ channels, respectively, reveal how such gating transitions
occur. Pore-lining "inner" helices contain a "gating hinge" that
bends by approximately 30 degrees. In a straight conformation,
four inner helices form a bundle, closing the pore near its
intracellular surface. In a bent configuration, the inner helices
splay open creating a wide (12 angstrom) entryway. Amino-acid
sequence conservation suggests a common structural basis for
gating in a wide range of K+ channels, both ligand- and voltage-
gated. The open conformation favors high conduction by
compressing the membrane field to the selectivity filter, and
also permits large organic cations and inactivation peptides to
enter the pore from the intracellular solution.

References (abridged):

1. del Camino, D., Holmgren, M., Liu, Y. & Yellen, G. Blocker
protection in the pore of a voltage-gated K+ channel and its
structural implications. Nature 403, 321-325 (2000)

2. Liu, Y., Holmgren, M., Jurman, M. E. & Yellen, G. Gated access
to the pore of a voltage-dependent K+ channel. Neuron 19, 175-184
(1997)

3. Doyle, D. A. et al. The structure of the potassium channel:
molecular basis of K+ conduction and selectivity. Science 280,
69-77 (1998)

4. Zhou, Y., Morais-Cabral, J. H., Kaufman, A. & MacKinnon, R.
Chemistry of ion coordination and hydration revealed by a K+
channel-Fab complex at 2.0 � resolution. Nature 414, 43-48 (2001)

5. del Camino, D. & Yellen, G. Tight steric closure at the
intracellular activation gate of a voltage-gated K+ channel.
Neuron 32, 649-656 (2001)

Nature 2002 417:523

Related Background Brief:

BLOCKER PROTECTION IN THE PORE OF A VOLTAGE-GATED K+ CHANNEL AND
ITS STRUCTURAL IMPLICATIONS. The structure of the bacterial
potassium channel KcsA has provided a framework for understanding
the related voltage-gated potassium channels (Kv channels) that
are used for signaling in neurons. Opening and closing of these
Kv channels (gating) occurs at the intracellular entrance to the
pore, and this is also the site at which many open channel
blockers affect Kv channels. To learn more about the sites of
blocker binding and about the structure of the open Kv channel,
the authors investigated the ability of blockers to protect
against chemical modification of cysteines introduced at sites in
transmembrane segment S6, which contributes to the intracellular
entrance. Within the intracellular half of S6, the authors found
an abrupt cessation of protection for both large and small
blockers that is inconsistent with the narrow "inner pore" seen
in the KcsA structure. These and other results are most readily
explained by supposing that the structure of Kv channels differs
from that of the non-voltage-gated bacterial channel by the
introduction of a sharp bend in the inner (S6) helices. This bend
would occur at a Pro-X-Pro sequence that is highly conserved in
Kv channels, near the site of activation gating. D. del Camino et
al: Nature 2000 403:321.

Related Background Brief:

GATED ACCESS TO THE PORE OF A VOLTAGE-DEPENDENT K+ CHANNEL.
Voltage-activated K+ channels are integral membrane proteins that
open or close a K(+)-selective pore in response to changes in
transmembrane voltage. Although the S4 region of these channels
has been implicated as the voltage sensor, little is known about
how opening and closing of the pore is accomplished. The authors
report they explored the gating process by introducing cysteines
at various positions thought to lie in or near the pore of the
Shaker K+ channel, and by testing their ability to be chemically
modified. The authors found a series of positions in the S6
transmembrane region that react rapidly with water-soluble thiol
reagents in the open state but not the closed state. An open-
channel blocker can protect several of these cysteines, showing
that they lie in the ion-conducting pore. At two of these sites,
Cd2+ ions bind to the cysteines without affecting the energetics
of gating; at a third site, Cd2+ binding holds the channel open.
The authors suggest the results indicate that these channels open
and close by the movement of an intracellular gate, distinct from
the selectivity filter, that regulates access to the pore. Y. Liu
et al: Neuron 1997 19:175.

Related Background Brief:

STRUCTURE OF THE KCSA CHANNEL INTRACELLULAR GATE IN THE OPEN
STATE. Ion channels catalyze the selective transfer of ions
across the membrane in response to a variety of stimuli. These
channels are "gated" by control of the access of ions to a
centrally located water-filled pore. The crystal structure of the
Streptomyces lividans potassium channel (KcsA) has allowed a
molecular exploration of this mechanism. Electron paramagnetic
resonance (EPR) studies have uncovered significant conformational
changes at the intracellular end of the second transmembrane
helix (TM2) upon gating. The authors report they have used site-
directed spin labeling (SDSL) and EPR spectroscopy in an attempt
to quantify the structural rearrangements of the KcsA TM2 bundle
underlying the transition from the closed to the open state.
Under conditions favoring the closed and open conformations, 10
intersubunit distances were obtained across TM2 segments from
tandem dimer constructs. Analysis of these data suggests a
mechanism in which each TM2 helix tilts away from the permeation
pathway towards the membrane plane and rotates about its helical
axis, supporting a scissoring-type motion with a pivot point near
residues 107-108. These movements are accompanied by a large
increase in the diameter of the vestibule below the central
water-filled cavity. Y.S. Liu et al: Nat Struct Biol 2001 8:883.

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6. ON ACID-SENSITIVE ION CHANNELS IN THE PERIPHERAL NERVOUS
SYSTEM.

D. Alvarez de la Rosa et al (Yale University, US) discuss acid-
sensitive channels, the authors making the following points:

1) Acid-sensitive ion channels (ASICs) are channels activated by
external protons that belong to the larger family known as
degenerins/epithelial Na+ channel (1). In mammalian organisms,
six different proteins arise from four genes. ASIC1 (BNaC2) (2,
3) and ASIC1 (4) are spliced forms of the ASIC1 gene; they differ
in the first 172 amino acids. ASIC2a (BNaC1 or MDEG1) (2, 5) and
ASIC2b (MDEG2) are spliced forms of the ASIC2 gene; they differ
in the first 236 amino acids. ASIC2b does not induce current but,
with ASIC3, forms functional heteromultimeric channels. ASIC3
(DRASIC) is activated by protons but not ASIC4 (SPASIC).

2) Expression of the ASIC genes in sensory neurons and activation
by extracellular protons have suggested that they may participate
in nociception. On the other hand, the structural similarity
shared with the degenerins, which are involved in light-touch
sensitivity in Caenorhabditis elegans, has prompted postulation
of a role for the ASICs in mechanoperception. The mammalian ASIC2
gene was recently disrupted in mouse. Knockout mice did not
exhibit a distinct phenotype, but careful examination of the
fibers innervating the skin revealed that the low-threshold,
rapidly adapting, and, to a lesser degree, slowly adapting
mechanoreceptors showed reduced discharge frequency on
stimulation when compared with wild-type animals.

3) In summary: Acid-sensitive ion channels (ASIC) are proton-
gated ion channels expressed in neurons of the mammalian central
and peripheral nervous systems. The functional role of these
channels is still uncertain, but they have been proposed to
constitute mechanoreceptors and/or nociceptors. The authors
report they have raised specific antibodies for ASIC1, ASIC2,
ASIC3, and ASIC4 to examine the distribution of these proteins in
neurons from dorsal root ganglia (DRG) and to determine their
subcellular localization. Western blot analysis demonstrates that
all four ASIC proteins are expressed in DRG and sciatic nerve.
Immunohistochemical experiments and functional measurements of
unitary currents from the ASICs with the patch-clamp technique
indicate that ASIC1 localizes to the plasma membrane of small-,
medium-, and large-diameter cells, whereas ASIC2 and ASIC3 are
preferentially in medium to large cells. Neurons coexpressing
ASIC2 and ASIC3 form predominantly heteromeric ASIC2-3 channels.
Two spliced forms, ASIC2a and ASIC2b, colocalize in the same
population of DRG neurons. Within cells, the ASICs are present
mainly on the plasma membrane of the soma and cellular processes.
Functional studies indicate that the pH sensitivity for
inactivation of ASIC1 is much higher than the one for activation;
hence, increases in proton concentration will inactivate the
channel. The authors suggest these functional properties and
localization in DRG have profound implications for the putative
functional roles of ASICs in the nervous system.

References (abridged):

1.  Fyfe, G. K. , Quin, A. M. & Canessa, C. M. (1998) Semin.
Nephrol. 18, 138-151

2.  Garc¡a-A¤overos, J. , Derfler, B. , Neville-Golden, J. ,
Hyman, B. T. & Corey, D. P. (1997) Proc. Natl. Acad. Sci. USA 94,
1459-1464

3.  Waldmann, R. , Champigny, G. , Bassilana, F. , Heurteaux, C.
& Lazdunski, M. (1997) Nature (London) 386, 173-177

4.  Chen, C. , England, S. , Akopian, A. N. & Wood, J. N. (1998)
Proc. Natl. Acad. Sci. USA 95, 10240-10245

5.  Price, M. P. , Snyder, P. M. & Welsh, M. J. (1996) J. Biol.
Chem. 271, 7879-7882

Proc. Nat. Acad. Sci. 2002 99:2326

Related Background Brief:

A PROTON-GATED CATION CHANNEL INVOLVED IN ACID-SENSING. Acid-
sensing is associated with both nociception and taste
transduction. Stimulation of sensory neurons by acid is of
particular interest, because acidosis accompanies many painful
inflammatory and ischemic conditions. The pain caused by acids is
thought to be mediated by (H+)-gated cation channels present in
sensory neurons. The authors report they have now cloned a (H+)-
gated channel (ASIC, for acid-sensing ionic channel) that belongs
to the amiloride-sensitive Na+ channel/degenerin family of ion
channels. Heterologous expression of ASIC induces an amiloride-
sensitive cation (Na+ > Ca2+ > K+) channel which is transiently
activated by rapid extracellular acidification. The biophysical
and pharmacological properties of the ASIC channel closely match
the (H+)-gated cation channel described in sensory neurons. ASIC
is expressed in dorsal root ganglia and is also distributed
widely throughout the brain. ASIC appears to be the simplest of
ligand-gated channels. R. Waldmann et al: Nature 1997 386:173.

Related Background Brief:

A SENSORY NEURON-SPECIFIC, PROTON-GATED ION CHANNEL. Proton-gated
channels expressed by sensory neurons are of particular interest
because low pH causes pain. Two proton-gated channels, acid-
sensing ionic channel (ASIC) and dorsal root ASIC (DRASIC), that
are members of the amiloride-sensitive ENaC/degenerin family are
known to be expressed by sensory neurons. The authors describe
the cloning and characterization of an ASIC splice variant, ASIC-
beta, which contains a unique N-terminal 172 aa, as well as
unique 5' and 3' untranslated sequences. ASIC-beta, unlike ASIC
and DRASIC, is found only in a subset of small and large diameter
sensory neurons and is absent from sympathetic neurons or the
central nervous system. The patterns of expression of ASIC and
ASIC-beta transcripts in rat dorsal root ganglion neurons are
distinct. When expressed in COS-7 cells, ASIC-beta forms a
functional channel with electrophysiological properties distinct
from ASIC and DRASIC. The pH dependency and sensitivity to
amiloride of ASIC-beta is similar to that described for ASIC, but
unlike ASIC, the channel is not permeable to calcium, nor are
ASIC-beta-mediated currents inhibited by extracellular calcium.
The unique distribution of ASIC-beta suggests that it may play a
specialized role in sensory neuron function. C-C. Chen et al:
Proc. Nat. Acad. Sci. 1998 95:10240.

Related Background Brief:

A MODULATORY SUBUNIT OF ACID SENSING ION CHANNELS IN BRAIN AND
DORSAL ROOT GANGLION CELLS. MDEG1 is a cation channel expressed
in brain that belongs to the degenerin/epithelial Na+ channel
superfamily. It is activated by the same mutations which cause
neurodegeneration in Caenorhabditis elegans if present in the
degenerins DEG-1, MEC-4, and MEC-10. MDEG1 shares 67% sequence
identity with the recently cloned proton-gated cation channel
ASIC (acid sensing ion channel), a new member of the family which
is present in brain and in sensory neurons. The authors report
they have now identified MDEG1 as a proton-gated channel with
properties different from those of ASIC. MDEG1 requires more
acidic pH values for activation and has slower inactivation
kinetics. In addition, the authors have cloned from mouse and rat
brain a splice variant form of the MDEG1 channel which differs in
the first 236 amino acids, including the first transmembrane
region. This new membrane protein, which has been called MDEG2,
is expressed in both brain and sensory neurons. MDEG2 is
activated neither by mutations that bring neurodegeneration once
introduced in C. elegans degenerins nor by low pH. However, it
can associate both with MDEG1 and another recently cloned (H+)-
activated channel DRASIC to form heteropolymers which display
different kinetics, pH dependences, and ion selectivities. Of
particular interest is the subunit combination specific for
sensory neurons, MDEG2/DRASIC. In response to a drop in pH, it
gives rise to a biphasic current with a sustained current which
discriminates poorly between Na+ and K+, like the native (H+)-
gated current recorded in dorsal root ganglion cells. This
sustained current is thought to be required for the tonic
sensation of pain caused by acids. E. Lingueglia et al: J Biol
Chem 1997 272:29778.

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7. ION CHANNEL PROTEINS AND CALCIUM CHANNELS

The regulated permeabilities of the biological cell membrane to
various ions are important factors in a number of crucial
cellular mechanisms. In general, these permeabilities involve
specific ion-selective pores constructed of proteins, the pores
called "ion channels", with ion channels of different types
available for any one ion species. Evidence suggests that an ion
channel protein spans the membrane and has a central water-filled
pore open to both the intracellular and extracellular
compartments. On each side, the pore widens to form a vestibule,
with the restricted region within the plane of the membrane
containing an effective "gate" that can open or close to control
the passage of ions.

Ion channels are highly regulated, linked to key cellular
processes, and during the past two decades, an intensive effort
in many laboratories has led to identification of the proteins of
some ion channels, studies of the configuration of these
proteins, and an improved understanding of the complex events
associated with the passage of simple ions such as sodium,
potassium, calcium, and chloride into and out of biological
cells. A very powerful technique used in much of this work
involves genetic engineering of ion channels. The essential idea
is to isolate a DNA sequence that encodes the protein for a
particular ion channel, then transfect this DNA sequence into the
genome of a host cell type amenable to detailed electrical and
transport measurements. When the ion channel protein is expressed
in this host cell and becomes part of the host cell plasma
membrane, the various properties of the ion channel become open
to investigation. Although the results of such experiments must
be carefully interpreted, the ability to make specific and
discrete alterations in channel protein membrane structure has
led to important insights into the relation between the
structures of ion channel proteins and their control of ion
permeabilities.

Of the ions that diffuse back and forth across cell membranes,
calcium ions are of great importance in many physiological
processes. In biological cells, extracellular and intracellular
concentrations of calcium ion differ by several orders of
magnitude, and cells are therefore exposed to a steep calcium ion
gradient across their membranes. In general, the control of
cellular calcium ion is maintained by an elaborate system of
channels, exchangers, and pumps located both in the plasma
membrane and in intracellular membranes.

James W. Putney Jr. (National Institutes of Health, US) presents
a commentary on recent studies of a calcium ion channel (L. Yue
et al: Nature 2001 410:705), with the author (Putney) making the
following points:

1) The author points out that calcium ions are important
biological signals, controlling processes such as protein
secretion, muscle contraction, cell death, and tissue
development. In general, calcium signaling involves an increase
in the intracellular concentration of calcium ions, and one of
the mechanisms by which this occurs is so-called "capacitative
calcium entry" (also called "store-operated calcium entry"), a
process that requires the regulated opening of ion channels in
the plasma membrane. These ion channels, however, have not yet
been identified. (The process is called "store-operated calcium
entry because it is somehow activated by a fall in the
concentration of calcium ions stored in an internal membrane
system, the endoplasmic reticulum.)

2) Yue et al now present evidence that implicates a newly
discovered protein (CaT1) as a constituent of a capacitative
calcium-entry channel, and the author (Putney) suggests this
discovery may lead to an improved understanding of the cellular
and molecular mechanism by which this channel is controlled.

3) The author (Putney) points out that although the weight of
evidence supports the conclusion of Yue et al that the protein
CaT1 (or, in some instances, a closely related protein [ECaC])
constitutes the ion-conducting pore of the calcium channel they
investigated, there are also other calcium-specific channels,
known from electrophysiological studies, that have properties
distinct from the channel investigated by Yue et al. Putney
points out that it is possible and even likely that other channel
proteins form part of these channels. Putney concludes: "In the
near future, I anticipate continuing progress in the search for
the complete molecular definition of the capacitative calcium-
entry channels, as well as a solution to the mystery of how they
are regulated."

Nature 2001 410:648

Related Background:

MOLECULAR CHARACTERIZATION OF A NEURONAL CALCIUM CHANNEL

The term "T-type channels" refers to channels whose ion currents
are both transient (due to rapid inactivation) and small (due to
small conductance), and such ion channels are believed to be
involved in pacemaker activity, low-threshold calcium ion spikes,
neuronal oscillations, etc. Frog oocytes are frog egg cells, and
they are a common laboratory vehicle for expressing the proteins
of genetically engineered material from other species and
coupling this expression with electrophysiological measurements
of frog oocyte membrane behavior.

Perez-Reyes et al (9 authors at 4 installations, US UK) report
the identification via cloning methods of a neuronal T-type
calcium ion channel, with expression of the protein constituting
the channel in frog oocytes, and electrophysiological
characterization of the channel in these cells. The authors
suggest they have cloned the first member of the low-voltage-
activated T-type Ca(sup2+) family, and one with identified human
and mouse genetic homologues.

Nature 1998 391:896

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8. ARTIFICIAL MEMBRANE RECEPTORS AND ION CHANNELS

S. Futaki et al (Kyoto University, JP) discuss artificial
receptors and ion channels, the authors making the following
points:

1) Natural receptor proteins and ion channel proteins serve in
the transduction of biological signals across cell membranes.
These molecules are often constructed by the association of
multiple homologous subunits to yield an organized structure.
Interaction of a specific ligand may then induce a conformational
switch to transmit specific ions in the cells as biological
signals.

2) Preserving the features of natural receptor proteins in
simplified peptide-based systems is a challenge in peptide
engineering offering potential for creating novel molecular
devices and channel protein models. Amphiphilic helical peptides
derived from transmembrane segments of natural ion channel
proteins and from artificial design have been shown to self-
assemble in membranes to form channels. For example, the assembly
of a mere approximately 20-amino-acid-residue peptide can
manifest a fundamental function of ion channel proteins, even
though the natural proteins are often composed of more than 1000
amino acids.

3) Self-assembly of channel peptides often produces a channel of
multiple open states, where the difference in the association
number (or the association state) may be detected as a difference
in channel conductance levels. To control the association number
of channel peptides, template molecules have been effectively
employed. The application of natural pore-forming membrane
proteins and non-peptide based molecules as frameworks for the
construction of artificial ion channels is potentially a means
for making designed channel pores, as well as a means to create
ion channels in which the ion flux can be controlled by external
stimuli such as binding of specific ligands and changes in the
electrical transmembrane potential.

4) The authors describe an experimental model ion channel system
involving the peptide antibiotic alamethicin with extra-membrane
control of channel peptide assembly, and the system exhibiting
channel current modulation.

J. Am. Chem. Soc. 2001 123:12127

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9. ION CHANNELS, SYNAPTIC TRANSMISSION, AND LONG-TERM
POTENTIATION

At the present time, the concept of "synaptic plasticity"
underlies nearly all theories of memories, the term referring to
changes in the behavior of the junction (synapse) between two
nerve cells resulting from past history. Two prominent aspects of
synaptic plasticity considered to be related to memory are
"facilitation" and "potentiation". The term "facilitation" refers
to a progressive increase in the amount of *neurotransmitter
substance released at a synapse by successive nerve impulses
(action potentials), the increase occurring during an input
barrage consisting of repetitive stimulation (stimulus train).
The term "potentiation" refers to an increase in neurotransmitter
substance released by an action potential following repetitive
stimulation of a synapse. Both facilitation and potentiation can
be long-lasting, and "long-term potentiation" has been a focus of
much research on the cellular basis of memory, particularly in
the hippocampus, a brain cortex structure in the medial part of
the temporal lobe. In humans, among other functions, the
hippocampus is apparently involved in short-term memory, and
analysis of the neurological correlates of learning behavior in
the rat indicates that the hippocampus of the rat is also
involved in memory.

V. Chevaleyre and P.E. Castillo (Albert Einstein College of
Medicine, US) discuss Ih channels, the authors making the
following points:

1) Voltage-dependent ion channels localized in presynaptic
terminals are ideally suited for modulating transmitter release.
Hyperpolarization -activated nonselective cationic channels, also
known as If, Iq, or Ih channels, are widely distributed in the
nervous system (1), and have been recently identified at
different presynaptic terminals such as the crustacean
neuromuscular junction (2), avian ciliary ganglion (3), the
basket cell synapses on Purkinje cells in the cerebellum (4), and
the calyx of Held in the brainstem (5). Although Ih channels are
thought to have diverse functions in neuronal regulation (1),
their contribution to neurotransmitter release is not fully
understood.

2) Ih, first identified and characterized in the heart, is a
noninactivating inward cation current carried by Na+/K+ (-30 to -
50 mV reversal potential) that slowly activates during
hyperpolarization. Because of these functional properties, Ih
channels have been postulated to contribute to the resting
membrane potential (as a fraction of these channels is open at
this potential), and control rhythmic activity in spontaneously
active cells. In some neurons, like hippocampal CA1 pyramidal
cells, Ih channels are highly expressed in the dendrites and
participate in regulating cable properties and temporal summation
of excitatory postsynaptic potential (EPSPs, ref. 5). Four genes
differentially expressed throughout the brain (termed HCN1-4 for
hyperpolarization-activated cyclic nucleotide-gated channels)
encode for products that form the Ih channels that display
different activation kinetics. One interesting property of these
channels is their strong regulation by cyclic nucleotides; i.e.,
cAMP positively modulates Ih channels by a change in the voltage-
dependence of channel activation. Targeting Ih channels, cyclic
nucleotides have been shown to
play a key role in regulating neuronal excitability and rhythmic
activity in the central nervous system.

3) Ih channels have received much attention lately because of the
idea that they may also modulate synaptic transmission and
presynaptic forms of plasticity. In the crayfish neuromuscular
junction for example, it has recently been postulated that
presynaptic Ih, by means of cAMP modulation, enhances transmitter
release by increasing the readily releasable vesicle pool (2).
However, in two different mammalian synapses in the brain where
Ih is present at the presynaptic terminal, no role of Ih channels
in basal synaptic transmission has been identified (4, 5),
casting doubt on the relevance of Ih in transmitter release. More
recently, Mellor et al. (2002) put forward the provocative idea
that presynaptic Ih channels are necessary for hippocampal mossy
fiber long-term potentiation (LTP). This form of plasticity is
expressed presynaptically and requires cAMP/protein kinase A
activation, and Ih channels are therefore excellent candidates
for underlying mossy fiber LTP because of their means of
modulation and possible presynaptic localization. Thus, LTP
expression could be caused by an Ih-mediated persistent
presynaptic depolarization resulting in a global change in
excitability.

References (abridged):

1. Pape, H. C. (1996) Annu. Rev. Physiol. 58, 299-327

2.  Beaumont, V. & Zucker, R. S. (2000) Nat. Neurosci. 3, 133-141

3.  Fletcher, G. H. & Chiappinelli, V. A. (1992) Brain Res. 575,
103-112

4. Southan, A. P. , Morris, N. P. , Stephens, G. J. & Robertson,
B. (2000) J. Physiol. 526, 91-97

5. Cuttle, M. F. , Rusznak, Z. , Wong, A. Y. , Owens, S. &
Forsythe, I. D. (2001) J. Physiol. 534, 733-744

Proc. Nat. Acad. Sci. 2002 99:9538

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