Potassium channel Kv1.2, structure in a membrane-like environment. Calculated hydrocarbon boundaries of the lipid bilayer are indicated by red and blue lines.
Potassium channels are the most widely distributed type of ion channel found in virtually all organisms. They form potassium-selective pores that span cell membranes. Potassium channels are found in most cell types and control a wide variety of cell functions.
Function
Potassium channels function to conduct potassium ions down their electrochemical gradient, doing so both rapidly (up to the diffusion rate of K+ ions in bulk water) and selectively (excluding, most notably, sodium despite the sub-angstrom difference in ionic radius). Biologically, these channels act to set or reset the resting potential in many cells. In excitable cells, such as neurons, the delayed counterflow of potassium ions shapes the action potential.
They also regulate cellular processes such as the secretion of hormones (e.g., insulin release from beta-cells in the pancreas) so their malfunction can lead to diseases (such as diabetes).
Some toxins, such as dendrotoxin, are potent because they block potassium channels.
Types
There are four major classes of potassium channels:
Tandem pore domain potassium channel
- are constitutively open or possess high basal activation, such as the
"resting potassium channels" or "leak channels" that set the negative
membrane potential of neurons.
The following table contains a comparison of the major classes of
potassium channels with representative examples (for a complete list of
channels within each class, see the respective class pages).
Top view of a potassium channel with potassium ions (purple) moving through the pore (in the center). (PDB: 1BL8)
Potassium channels have a tetrameric structure in which four identical protein subunits associate to form a fourfold symmetric (C4)
complex arranged around a central ion conducting pore (i.e., a
homotetramer). Alternatively four related but not identical protein
subunits may associate to form heterotetrameric complexes with pseudo C4
symmetry. All potassium channel subunits have a distinctive pore-loop
structure that lines the top of the pore and is responsible for
potassium selective permeability.
There are over 80 mammaliangenes that encode potassium channel subunits. However potassium channels found in bacteria are amongst the most studied of ion channels, in terms of their molecular structure. Using X-ray crystallography,[55][56] profound insights have been gained into how potassium ions pass through these channels and why (smaller) sodium ions do not.[57] The 2003 Nobel Prize for Chemistry was awarded to Rod MacKinnon for his pioneering work in this area.[58]
Selectivity filter
Crystallographic structure of the bacterial KcsA potassium channel (PDB: 1K4C).
In this figure, only two of the four subunits of the tetramer are
displayed for the sake of clarity. The protein is displayed as a green
cartoon diagram. In addition backbone carbonyl groups and threonine
sidechain protein atoms (oxygen = red, carbon = green) are displayed.
Finally potassium ions (occupying the S2 and S4 sites) and the oxygen
atoms of water molecules (S1 and S3) are depicted as purple and red
spheres respectively.
Potassium ion channels remove the hydration shell from the ion when
it enters the selectivity filter. The selectivity filter is formed by a
five residue sequence, TVGYG, termed the signature sequence, within each
of the four subunits. This signature sequence is within a loop between
the pore helix and TM2/6, historically termed the P-loop. This signature
sequence is highly conserved, with the exception that a valine residue
in prokaryotic potassium channels is often substituted with an
isoleucine residue in eukaryotic channels. This sequence adopts a unique
main chain structure, structurally analogous to a nest protein structural motif. The four sets of electronegativecarbonyl oxygen atoms
are aligned toward the center of the filter pore and form a square
antiprism similar to a water-solvating shell around each potassium
binding site. The distance between the carbonyl oxygens and potassium
ions in the binding sites of the selectivity filter is the same as
between water oxygens in the first hydration shell and a potassium ion
in water solution, providing an energetically-favorable route for de-solvation
of the ions. Sodium ions, however, are too small to fill the space
between the carbonyl oxygen atoms. Thus, it is energetically favorable
for sodium ions to remain bound with water molecules in the
extracellular space, rather than to pass through the potassium-selective
ion pore. This width appears to be maintained by hydrogen bonding and van der Waals forces within a sheet of aromatic amino acid residues surrounding the selectivity filter. The selectivity filter opens towards the extracellular solution, exposing four carbonyl oxygens in a glycine residue (Gly79 in KcsA).
The next residue toward the extracellular side of the protein is the
negatively charged Asp80 (KcsA). This residue together with the five
filter residues form the pore that connects the water-filled cavity in
the center of the protein with the extracellular solution.
Selectivity mechanism
The
mechanism of potassium channel selectivity remains under continued
debate. The carbonyl oxygens are strongly electro-negative and
cation-attractive. The filter can accommodate potassium ions at 4 sites
usually labelled S1 to S4 starting at the extracellular side. In
addition, one ion can bind in the cavity at a site called SC or one or
more ions at the extracellular side at more or less well-defined sites
called S0 or Sext. Several different occupancies of these sites are
possible. Since the X-ray structures are averages over many molecules,
it is, however, not possible to deduce the actual occupancies directly
from such a structure. In general, there is some disadvantage due to
electrostatic repulsion to have two neighboring sites occupied by ions.
Proposals for the mechanism of selectivity have been made based on molecular dynamics simulations, toy models of ion binding, thermodynamic calculations, topological considerations, and structural differences between selective and non-selective channels.
The mechanism for ion translocation in KcsA has been studied extensively by theoretical calculations and simulation.
The prediction of an ion conduction mechanism in which the two doubly
occupied states (S1, S3) and (S2, S4) play an essential role has been
affirmed by both techniques. Molecular dynamics (MD) simulations suggest the two extracellular states, Sext and S0, reflecting ions entering and leaving the filter, also are important actors in ion conduction.
Hydrophobic region
This
region neutralizes the environment around the potassium ion so that it
is not attracted to any charges. In turn, it speeds up the reaction.
Central cavity
A central pore, 10 Å wide, is located near the center of the transmembrane channel, where the energy barrier
is highest for the transversing ion due to the hydrophobity of the
channel wall. The water-filled cavity and the polar C-terminus of the
pore helices ease the energetic barrier for the ion. Repulsion by
preceding multiple potassium ions is thought to aid the throughput of
the ions.
The presence of the cavity can be understood intuitively as one of the
channel's mechanisms for overcoming the dielectric barrier, or repulsion
by the low-dielectric membrane, by keeping the K+ ion in a watery, high-dielectric environment.
Regulation
Graphical representation of open and shut potassium channels (PDB: 1lnq and PDB: 1k4c).
Two simple bacterial channels are shown to compare the "open" channel
structure on the right with the "closed" structure on the left. At top
is the filter (selects potassium ions), and at bottom is the gating
domain (controls opening and closing of channel).
The flux of ions through the potassium channel pore is regulated by two related processes, termed gating
and inactivation. Gating is the opening or closing of the channel in
response to stimuli, while inactivation is the rapid cessation of
current from an open potassium channel and the suppression of the
channel's ability to resume conducting. While both processes serve to
regulate channel conductance, each process may be mediated by a number
of mechanisms.
Generally, gating is thought to be mediated by additional
structural domains which sense stimuli and in turn open the channel
pore. These domains include the RCK domains of BK channels, and voltage sensor domains of voltage gated K+
channels. These domains are thought to respond to the stimuli by
physically opening the intracellular gate of the pore domain, thereby
allowing potassium ions to traverse the membrane. Some channels have
multiple regulatory domains or accessory proteins, which can act to
modulate the response to stimulus. While the mechanisms continue to be
debated, there are known structures of a number of these regulatory
domains, including RCK domains of prokaryoticand eukaryotic channels, pH gating domain of KcsA, cyclic nucleotide gating domains, and voltage gated potassium channels.
N-type inactivation is typically the faster inactivation mechanism, and is termed the "ball and chain" model.
N-type inactivation involves interaction of the N-terminus of the
channel, or an associated protein, which interacts with the pore domain
and occludes the ion conduction pathway like a "ball". Alternatively,
C-type inactivation is thought to occur within the selectivity filter
itself, where structural changes within the filter render it
non-conductive. There are a number of structural models of C-type
inactivated K+ channel filters, although the precise mechanism remains unclear.
Potassium channel blockers inhibit the flow of potassium ions through
the channel. They either compete with potassium binding within the
selectivity filter or bind outside the filter to occlude ion conduction.
An example of one of these competitors is quaternary ammonium ions,
which bind at the extracellular face or central cavity of the channel.
For blocking from the central cavity quaternary ammonium ions are also
known as open channel blockers, as binding classically requires the
prior opening of the cytoplasmic gate.
Barium ions can also block potassium channel currents, by binding with high affinity within the selectivity filter. This tight binding is thought to underlie barium toxicity by inhibiting potassium channel activity in excitable cells.
Medically potassium channel blockers, such as 4-aminopyridine and 3,4-diaminopyridine, have been investigated for the treatment of conditions such as multiple sclerosis. Off target drug effects can lead to drug induced Long QT syndrome, a potentially life-threatening condition. This is most frequently due to action on the hERG potassium channel in the heart. Accordingly, all new drugs are preclinically tested for cardiac safety.
Birth of an Idea (2007) by Julian Voss-Andreae. The sculpture was commissioned by Roderick MacKinnon based on the molecule's atomic coordinates that were determined by MacKinnon's group in 2001.
Some types of potassium channels are activated by muscarinic receptors and these are called muscarinic potassium channels (IKACh). These channels are a heterotetramer composed of two GIRK1 and two GIRK4 subunits. Examples are potassium channels in the heart, which, when activated by parasympathetic signals through M2 muscarinic receptors, cause an outward current of potassium, which slows down the heart rate.
In fine art
Roderick MacKinnon commissioned Birth of an Idea, a 5-foot (1.5 m) tall sculpture based on the KcsA potassium channel.
The artwork contains a wire object representing the channel's interior
with a blown glass object representing the main cavity of the channel
structure.
Micrograph showing amyloid deposits (pink) in small bowel.
Duodenum with amyloid deposition in lamina propria. Amyloid shows up as
homogeneous pink material in lamina propria and around blood vessels.
20× magnification.
Amyloids are aggregates of proteins characterised by a fibrillar morphology of typically 7–13 nm in diameter, a β-sheetsecondary structure (known as cross-β) and ability to be stained by particular dyes, such as Congo red. In the human body, amyloids have been linked to the development of various diseases. Pathogenic amyloids form when previously healthy proteins lose their normal structure and physiological functions (misfolding)
and form fibrous deposits within and around cells. These protein
misfolding and deposition processes disrupt the healthy function of
tissues and organs.
Such amyloids have been associated with (but not necessarily as the cause of) more than 50 human diseases, known as amyloidosis, and may play a role in some neurodegenerative diseases. Some of these diseases are mainly sporadic and only a few cases are familial. Others are only familial. Some result from medical treatment. Prions are an infectious form of amyloids that can act as a template to convert other non-infectious forms. Amyloids may also have normal biological functions; for example, in the formation of fimbriae in some genera of bacteria, transmission of epigenetic traits in fungi, as well as pigment deposition and hormone release in humans.
Amyloids have been known to arise from many different proteins. These polypeptide chains generally form β-sheet
structures that aggregate into long fibers; however, identical
polypeptides can fold into multiple distinct amyloid conformations. The diversity of the conformations may have led to different forms of the prion diseases.
An unusual secondary structure named α sheet has been proposed as the toxic constituent of amyloid precursor proteins, but this idea is not widely accepted at present.
Amyloid of HET-s(218-289) prion pentamer, Podospora anserina (PDB: 2rnm)
Definition
The name amyloid comes from the early mistaken identification by Rudolf Virchow of the substance as starch (amylum in Latin, from Ancient Greek: ἄμυλον, romanized: amylon),
based on crude iodine-staining techniques. For a period, the scientific
community debated whether or not amyloid deposits are fatty deposits or carbohydrate deposits until it was finally found (in 1859) that they are, in fact, deposits of albumoid proteinaceous material.
The classical, histopathological definition of amyloid is an extracellular, proteinaceous fibrillar deposit exhibiting β-sheetsecondary structure and identified by apple-green birefringence when stained with congo red under polarized light. These deposits often recruit various sugars and other components such as serum amyloid P component, resulting in complex, and sometimes inhomogeneous structures.
Recently this definition has come into question as some classic,
amyloid species have been observed in distinctly intracellular
locations.
To date, 37 human proteins have been found to form amyloid in pathology and be associated with well-defined diseases.
The International Society of Amyloidosis classifies amyloid fibrils and
their associated diseases based upon associated proteins (for example
ATTR is the group of diseases and associated fibrils formed by TTR). A table is included below.
Many
examples of non-pathological amyloid with a well-defined physiological
role have been identified in various organisms, including human. These may be termed as functional or physiological or native amyloid.
Curlifibrils produced by E. coli,Salmonella, and a few other members of the Enterobacteriales (Csg). The genetic elements (operons) encoding the curli system are phylogenetic widespread and can be found in at least four bacterial phyla. This suggest that many more bacteria may express curli fibrils.
GvpA, forming the walls of particular Gas vesicles, i.e. the buoyancy organelles of aquatic archaea and eubacteria
Several yeast prions are based on an infectious amyloid, e.g. [PSI+] (Sup35p); [URE3] (Ure2p); [PIN+] or [RNQ+] (Rnq1p); [SWI1+] (Swi1p) and [OCT8+] (Cyc8p)
Structure
of a fibril, consisting of one single protofilament, of the amyloid β
peptide viewed down the long axis of the fibril (PDB: 2mlq)
Amyloids are formed of long unbranched fibers that are characterized by an extended β-sheet secondary structure in which individual β strands
(β-strands) (coloured arrows in the adjacent figure) are arranged in an
orientation perpendicular to the long axis of the fiber. Such a
structure is known as cross-β structure. Each individual fiber may be
7–13 nanometres in width and a few micrometres in length. The main hallmarks recognised by different disciplines to classify
protein aggregates as amyloid is the presence of a fibrillar morphology
with the expected diameter, detected using transmission electron microscopy (TEM) or atomic force microscopy (AFM), the presence of a cross-β secondary structure, determined with circular dichroism, FTIR, solid-state nuclear magnetic resonance (ssNMR), X-ray crystallography, or X-ray fiber diffraction
(often considered the "gold-standard" test to see whether a structure
contains cross-β fibres), and an ability to stain with specific dyes,
such as Congo red, thioflavin T or thioflavin S.
The term "cross-β" was based on the observation of two sets of
diffraction lines, one longitudinal and one transverse, that form a
characteristic "cross" pattern. There are two characteristic scattering diffraction signals produced at 4.7 and 10 Å (0.47 nm and 1.0 nm), corresponding to the interstrand and stacking distances in β sheets.
The "stacks" of β sheet are short and traverse the breadth of the
amyloid fibril; the length of the amyloid fibril is built by aligned
β-strands. The cross-β pattern is considered a diagnostic hallmark of
amyloid structure.
Amyloid fibrils are generally composed of 1–8 protofilaments (one
protofilament also corresponding to a fibril is shown in the figure),
each 2–7 nm in diameter, that interact laterally as flat ribbons that
maintain the height of 2–7 nm (that of a single protofilament) and are
up to 30 nm wide; more often protofilaments twist around each other to
form the typically 7–13 nm wide fibrils.
Each protofilament possesses the typical cross-β structure and may be
formed by 1–6 β-sheets (six are shown in the figure) stacked on each
other. Each individual protein molecule can contribute one to several
β-strands in each protofilament and the strands can be arranged in
antiparallel β-sheets, but more often in parallel β-sheets. Only a
fraction of the polypeptide chain is in a β-strand conformation in the
fibrils, the remainder forms structured or unstructured loops or tails.
For a long time our knowledge of the atomic-level structure of
amyloid fibrils was limited by the fact that they are unsuitable for the
most traditional methods for studying protein structures. Recent years
have seen progress in experimental methods, including solid-state NMR spectroscopy and Cryo-Electron Microscopy.
Combined, these methods have provided 3D atomic structures of amyloid
fibrils formed by amyloid β peptides, α-synuclein, tau, and the FUS
protein, associated with various neurodegenerative diseases.
X-ray diffraction studies of microcrystals revealed atomistic
details of core region of amyloid, although only for simplified
peptides having a length remarkably shorter than that of peptides or
proteins involved in disease.
The crystallographic structures show that short stretches from
amyloid-prone regions of amyloidogenic proteins run perpendicular to the
filament axis, consistent with the "cross-β" feature of amyloid
structure. They also reveal a number of characteristics of amyloid
structures – neighboring β-sheets are tightly packed together via an
interface devoid of water (therefore referred to as dry interface), with
the opposing β-strands slightly offset from each other such that their
side-chains interdigitate. This compact dehydrated interface created was
termed a steric-zipper interface.
There are eight theoretical classes of steric-zipper interfaces,
dictated by the directionality of the β-sheets (parallel and
anti-parallel) and symmetry between adjacent β-sheets. A limitation of
X-ray crystallography for solving amyloid structure is represented by
the need to form microcrystals, which can be achieved only with peptides
shorter than those associated with disease.
Although bona fide amyloid structures always are based on
intermolecular β-sheets, different types of "higher order" tertiary
folds have been observed or proposed. The β-sheets may form a β-sandwich, or a β-solenoid which may be either β-helix
or β-roll. Native-like amyloid fibrils in which native β-sheet
containing proteins maintain their native-like structure in the fibrils
have also been proposed.
There are few developed ideas on how the complex backbone topologies of
disulfide-constrained proteins, which are prone to form amyloid fibrils
(such as insulin and lysozyme), adopt the amyloid β-sheet motif. The
presence of multiple constraints significantly reduces the accessible
conformational space, making computational simulations of amyloid
structures more feasible.
One complicating factor in studies of amyloidogenic polypeptides
is that identical polypeptides can fold into multiple distinct amyloid
conformations. This phenomenon is typically described as amyloid polymorphism. It has notable biological consequences given that it is thought to explain the prion strain phenomenon.
Amyloid is formed through the polymerization of hundreds to thousands of monomeric peptides or proteins into long fibers. Amyloid formation involves a lag phase (also called nucleation phase), an exponential phase (also called growth phase) and a plateau phase (also called saturation phase), as shown in the figure. Indeed, when the quantity of fibrils is plotted versus time, a sigmoidal time course is observed reflecting the three distinct phases.
In the simplest model of 'nucleated polymerization' (marked by
red arrows in the figure below), individual unfolded or partially
unfolded polypeptide chains (monomers) convert into a nucleus (monomer or oligomer) via a thermodynamically unfavourable process that occurs early in the lag phase. Fibrils grow subsequently from these nuclei through the addition of monomers in the exponential phase.
A different model, called 'nucleated conformational conversion'
and marked by blue arrows in the figure below, was introduced later on
to fit some experimental observations: monomers have often been found to
convert rapidly into misfolded and highly disorganized oligomers
distinct from nuclei.
Only later on, will these aggregates reorganise structurally into
nuclei, on which other disorganised oligomers will add and reorganise
through a templating or induced-fit mechanism (this 'nucleated
conformational conversion' model), eventually forming fibrils.
Normally folded proteins have to unfold partially before aggregation can take place through one of these mechanisms. In some cases, however, folded proteins can aggregate without crossing the major energy barrier for unfolding, by populating native-like conformations as a consequence of thermal fluctuations, ligand release or local unfolding occurring in particular circumstances.
In these native-like conformations, segments that are normally buried
or structured in the fully folded and possessing a high propensity to
aggregate become exposed to the solvent or flexible, allowing the
formation of native-like aggregates, which convert subsequently into
nuclei and fibrils. This process is called 'native-like aggregation'
(green arrows in the figure) and is similar to the 'nucleated
conformational conversion' model.
A more recent, modern and thorough model of amyloid fibril
formation involves the intervention of secondary events, such as
'fragmentation', in which a fibril breaks into two or more shorter
fibrils, and 'secondary nucleation', in which fibril surfaces (not
fibril ends) catalyze the formation of new nuclei.
Both secondary events increase the number of fibril ends able to
recruit new monomers or oligomers, therefore accelerating fibril
formation through a positive feedback mechanism. These events add to the
well recognised steps of primary nucleation (formation of the nucleus
from the monomers through one of models described above), fibril
elongation (addition of monomers or oligomers to growing fibril ends)
and dissociation (opposite process).
Such a new model is described in the figure on the right and involves the utilization of a master equation
that includes all steps of amyloid fibril formation, i.e. primary
nucleation, fibril elongation, secondary nucleation and fibril
fragmentation. The rate constants of the various steps can be determined from a global fit of a number of time courses of aggregation (for example ThT fluorescence emission versus time) recorded at different protein concentrations. The general master equation approach to amyloid fibril formation with secondary pathways has been developed by Knowles, Vendruscolo, Cohen, Michaels and coworkers and considers the time evolution of the concentration of fibrils of length (here represents the number of monomers in an aggregate). where denotes the Kronecker delta.
The physical interpretation of the various terms in the above master
equation is straight forward: the terms on the first line describe the
growth of fibrils via monomer addition with rate constant (elongation). The terms on the second line describe monomer dissociation, i.e. the inverse process of elongation.
is the rate constant of monomer dissociation. The terms on the third
line describe the effect of fragmentation, which is assumed to occur
homogeneously along fibrils with rate constant .
Finally, the terms on the last line describe primary and secondary
nucleation respectively. Note that the rate of secondary nucleation is
proportional to the mass of aggregates, defined as .
Following this analytical approach, it has become apparent that
the lag phase does not correspond necessarily to only nucleus formation,
but rather results from a combination of various steps. Similarly, the
exponential phase is not only fibril elongation, but results from a
combination of various steps, involving primary nucleation, fibril
elongation, but also secondary events. A significant quantity of fibrils
resulting from primary nucleation and fibril elongation may be formed
during the lag phase and secondary steps, rather than only fibril
elongation, can be the dominant processes contributing to fibril growth
during the exponential phase. With this new model, any perturbing agents
of amyloid fibril formation, such as putative drugs, metabolites, mutations, chaperones, etc., can be assigned to a specific step of fibril formation.
Amino acid sequence and amyloid formation
In general, amyloid polymerization
(aggregation or non-covalent polymerization) is sequence-sensitive,
that is mutations in the sequence can induce or prevent self-assembly.For example, humans produce amylin,
an amyloidogenic peptide associated with type II diabetes, but in rats
and mice prolines are substituted in critical locations and
amyloidogenesis does not occur. Studies comparing synthetic to recombinant β amyloid peptide
in assays measuring rate of fibrillation, fibril homogeneity, and
cellular toxicity showed that recombinant β amyloid peptide has a faster
fibrillation rate and greater toxicity than synthetic β amyloid
peptide.
There are multiple classes of amyloid-forming polypeptide sequences. Glutamine-rich polypeptides are important in the amyloidogenesis of Yeast and mammalian prions, as well as trinucleotide repeat disorders including Huntington's disease.
When glutamine-rich polypeptides are in a β-sheet conformation,
glutamines can brace the structure by forming inter-strand hydrogen
bonding between its amide carbonyls and nitrogens of both the backbone
and side chains. The onset age for Huntington's disease shows an inverse
correlation with the length of the polyglutamine sequence, with analogous findings in a C. elegans model system with engineered polyglutamine peptides.
Other polypeptides and proteins such as amylin
and the β amyloid peptide do not have a simple consensus sequence and
are thought to aggregate through the sequence segments enriched with
hydrophobic residues, or residues with high propensity to form β-sheet
structure. Among the hydrophobic residues, aromatic amino-acids are found to have the highest amyloidogenic propensity.
Cross-polymerization (fibrils of one polypeptide sequence causing
other fibrils of another sequence to form) is observed in vitro and
possibly in vivo. This phenomenon is important, since it would explain
interspecies prion propagation and differential rates of prion propagation, as well as a statistical link between Alzheimer's and type 2 diabetes.
In general, the more similar the peptide sequence the more efficient
cross-polymerization is, though entirely dissimilar sequences can
cross-polymerize and highly similar sequences can even be "blockers"
that prevent polymerization.
Amyloid toxicity
The
reasons why amyloid cause diseases are unclear. In some cases, the
deposits physically disrupt tissue architecture, suggesting disruption
of function by some bulk process. An emerging consensus implicates
prefibrillar intermediates, rather than mature amyloid fibers, in
causing cell death, particularly in neurodegenerative diseases.[17][69]
The fibrils are, however, far from innocuous, as they keep the protein
homeostasis network engaged, release oligomers, cause the formation of
toxic oligomers via secondary nucleation, grow indefinitely spreading
from district to district[2] and, in some cases, may be toxic themselves.[70]
Calcium dysregulation has been observed to occur early in cells
exposed to protein oligomers. These small aggregates can form ion
channels through lipid bilayer membranes and activate NMDA and AMPA
receptors. Channel formation has been hypothesized to account for
calcium dysregulation and mitochondrial dysfunction by allowing
indiscriminate leakage of ions across cell membranes.[71] Studies have shown that amyloid deposition is associated with mitochondrial dysfunction and a resulting generation of reactive oxygen species (ROS), which can initiate a signalling pathway leading to apoptosis.[72]
There are reports that indicate amyloid polymers (such as those of
huntingtin, associated with Huntington's disease) can induce the
polymerization of essential amyloidogenic proteins, which should be
deleterious to cells. Also, interaction partners of these essential
proteins can also be sequestered.[73]
All these mechanisms of toxicity are likely to play a role. In
fact, the aggregation of a protein generates a variety of aggregates,
all of which are likely to be toxic to some degree. A wide variety of
biochemical, physiological and cytological perturbations has been
identified following the exposure of cells and animals to such species,
independently of their identity. The oligomers have also been reported
to interact with a variety of molecular targets. Hence, it is unlikely
that there is a unique mechanism of toxicity or a unique cascade of
cellular events. The misfolded nature of protein aggregates causes a
multitude of aberrant interactions with a multitude of cellular
components, including membranes, protein receptors, soluble proteins,
RNAs, small metabolites, etc.
Histological staining
In the clinical setting, amyloid diseases are typically identified by a change in the spectroscopic properties of planar aromaticdyes such as thioflavin T, congo red or NIAD-4. In general, this is attributed to the environmental change, as these dyes intercalate between β-strands to confine their structure.
Congo Red positivity remains the gold standard for diagnosis of amyloidosis. In general, binding of Congo Red to amyloid plaques produces a typical apple-green birefringence
when viewed under cross-polarized light. Recently, significant
enhancement of fluorescence quantum yield of NIAD-4 was exploited to super-resolution fluorescence imaging of amyloid fibrils and oligomers. To avoid nonspecific staining, other histology stains, such as the hematoxylin and eosin
stain, are used to quench the dyes' activity in other places such as
the nucleus, where the dye might bind. Modern antibody technology and immunohistochemistry
has made specific staining easier, but often this can cause trouble
because epitopes can be concealed in the amyloid fold; in general, an
amyloid protein structure is a different conformation from the one that
the antibody recognizes.
