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Proc Natl Acad Sci U S A. 1999 July 6; 96(14): 7658–7663. | PMCID: PMC33597 |
Copyright © 1999, The National Academy of Sciences Ion channels gated by heat P. Cesare, A. Moriondo, V. Vellani, and P. A. McNaughton* Neuroscience Research Centre, King’s College London Strand, London WC2R 2LS, United Kingdom All animals need to sense temperature to avoid hostile environments
and to regulate their internal homeostasis. A particularly obvious
example is that animals need to avoid damagingly hot stimuli. The
mechanisms by which temperature is sensed have until recently been
mysterious, but in the last couple of years, we have begun to
understand how noxious thermal stimuli are detected by sensory neurons.
Heat has been found to open a nonselective cation channel in primary
sensory neurons, probably by a direct action. In a separate study, an
ion channel gated by capsaicin, the active ingredient of chili peppers,
was cloned from sensory neurons. This channel (vanilloid receptor
subtype 1, VR1) is gated by heat in a manner similar to the native
heat-activated channel, and our current best guess is that this channel
is the molecular substrate for the detection of painful heat. Both the
heat channel and VR1 are modulated in interesting ways. The response of
the heat channel is potentiated by phosphorylation by protein kinase C,
whereas VR1 is potentiated by externally applied protons. Protein
kinase C is known to be activated by a variety of inflammatory
mediators, including bradykinin, whereas extracellular acidification is
characteristically produced by anoxia and inflammation. Both modulatory
pathways are likely, therefore, to have important physiological
correlates in terms of the enhanced pain (hyperalgesia) produced by
tissue damage and inflammation. Future work should focus on
establishing, in molecular terms, how a single ion channel can detect
heat and how the detection threshold can be modulated by hyperalgesic
stimuli. Organisms sense temperature for all sorts of reasons. Highly
accurate thermosensation is required to set the body temperature of a
mammal. Simpler animals sense the external temperature to seek out
favorable environments for feeding or for mating. Damaging extremes of
temperature must be avoided, of course, and for this purpose,
pain-sensitive nerve terminals detect very low and very high
temperatures and induce an avoidance response. In all these instances,
temperature must be detected—but how? In many cases, the detection
mechanism involves a specialized temperature-sensitive nerve terminal,
which, on application of a temperature change, generates a
depolarization and a resulting train of action potentials in the
sensory nerve axon. So it is perhaps obvious to state that temperature
must gate an ion channel in the sensory nerve terminal. But how does it
work? One could imagine a temperature-sensitive biochemical pathway
that modulates an internal transmitter and in turn gates the ion
channel. There is some evidence for such a system in at least one
temperature-sensitive pathway in the nematode
Caenorhabditis elegans (see below). But in
the only other instance of which we have any understanding, the action
of temperature on the ion channel seems instead to be direct. To the
existing voltage-gated, ligand-gated, and mechanosensitive ion
channels, we can therefore add a fourth major category of ion channels,
namely, heat-sensitive ion channels. This article reviews our
understanding to date of this newly characterized class of ion
channels. Heat-Sensitive Ion Channels in Primary Sensory Neurons. The
most direct way to study the detection of hot stimuli is in
situ, either by asking subjects at what temperature a sensation of
warmth changes to a sensation of pain or alternatively by recording the
frequency of action potentials in the axon of a primary pain-sensitive
neuron (a nociceptor) while a thermal stimulus is applied to the
receptive field. Experiments like these have shown that, as the
temperature is raised, a sensation of warmth changes to pain at around
43–45°C and that the intensity of the pain sensation increases
steeply thereafter ( 1). Recordings of action potentials from
nociceptive nerve fibers show a similar picture, namely of a threshold
for initiation of action potentials at 43–45°C and a steep increase
in firing frequency as the temperature is increased further ( 1,
2). To take things much further, for instance to study the pathways
controlling ionic currents involved in the transduction process, a
preparation of isolated nociceptors is needed. Other sensory receptors
can be isolated more or less intact, and the study of isolated
photoreceptors, auditory receptors, olfactory receptors, etc. has told
us a great deal about their mechanisms of operation. Nociceptors are
unfortunately a much more difficult proposition. The sensory terminals
are extremely fine and are embedded in a cellular matrix whose
disruption during dissection releases the very signaling molecules that
the nociceptor nerve terminal is designed to detect. The difficulty of
isolating intact nociceptive nerve terminals has meant that studies on
isolated nociceptors have all been on neuronal cell bodies. In a
typical procedure, the neuronal cell body is isolated by enzymatic
treatment and is cultured for a few days before use ( 3, 4). The sensory
terminals are, of course, completely removed during the isolation
procedure, and we must hope that the properties of those terminals are
recreated in the cultured cell body and dendrites. When they are
acutely isolated, nociceptive cell bodies often fail to respond to
noxious stimuli, but in a process that is poorly understood,
nociceptive properties characteristic of the sensory terminal reappear
after a few days in culture in the presence of serum and nerve growth
factor ( 3– 6). This preparation of cultured nociceptors has been used
for almost all experiments investigating the cellular and molecular
basis of detection of painful stimuli. The complexity of the procedure
for isolating nociceptors nonetheless makes it essential that we check
very carefully that our nociceptors’ responses resemble those in
vivo. An example of the response of the membrane potential of a cultured
nociceptor to application of a 49°C heat stimulus is shown in Fig.
( 6, 7). As is seen in
nociceptive nerve terminals in vivo ( 8), the heat stimulus
causes a rapid depolarization and initiates a train of action
potentials. The fact that this response is present in isolated
nociceptors shows that no other cells are necessary to produce the
response to heat; there is no signal molecule released from adjacent
damaged cells to which the nociceptor responds. Nor is the response due
to damage to the nociceptive neuron itself—as might occur if heat were
causing a breakdown in the plasma membrane and a consequent
depolarization—because the depolarization and action-potential firing
ceases immediately when the stimulus is withdrawn and because similar
responses can be elicited again and again on repeated application of
the heat stimulus.
The reproducibility of the response in isolated nociceptors resembles
that in other sensory receptors. Interestingly, though, the behavior of
nociceptors in vivo is different. Repeated application of a
strong stimulus leads to a progressive increase in the response in
nociceptors in vivo but not in isolated nociceptors nor in
other sensory receptors. This process, known as sensitization or
hyperalgesia, is characteristic of nociceptors in vivo and
has obvious protective value for the organism as a whole, in that the
pain caused by a damaging stimulus becomes more urgent if the stimulus
is repeated or maintained. The fact that sensitization is not observed
in isolated nociceptors suggests that the phenomenon is not intrinsic
to the neuron but instead has its origin in extracellular signals
released from nearby damaged or inflamed tissue ( 9). Recent advances in
our understanding of this process of sensitization are discussed below. The membrane current induced by heat in a voltage-clamped nociceptor is
shown in Fig. A. The
current is activated rapidly (but not instantaneously) by heat, with a
mean time to half activation of ≈35 ms at 50°C ( 6, 7). By contrast,
neurons insensitive to heat show only a small current change, which
occurs as rapidly as the solution change and therefore probably has a
simple physical origin such as a temperature dependence of membrane
leakage resistance. When the temperature dependence of the
heat-sensitive current is examined (Fig. B), the
current can be seen to be activated above ≈42°C and to increase
exponentially as the temperature is raised further, much as is observed
in nociceptors in vivo ( 1, 2).
Experiments examining the ionic selectivity of the heat-activated
current ( 6, 7) have shown that the heat-activated channel discriminates
poorly amongst monovalent alkali cations, in common with many other ion
channels such as those gated by glutamate, acetylcholine, or cyclic
nucleotides. Calcium ions can by themselves carry current through the
channel but, in addition, have the effect of partially blocking a
current carried by monovalent ions. The channel must therefore possess
a binding site in the pore region with a higher affinity for
Ca 2+ than for monovalent cations. Contrary to
early reports ( 10), the channel does not seem to be blocked by
Cs + ions. The current-voltage relation shows
outward rectification and a reversal potential of around 0 mV under
physiological conditions ( 6, 10, 11). Single heat-activated channels have a conductance of around 30–40 pS
( 10, 11). The single channel conductance itself is only weakly
temperature-dependent, in common with other ion channels, and the
pronounced dependence of current on temperature is caused by a strong
temperature dependence of the probability of channel opening. The time
constants of channel opening can be deduced from the characteristics of
the current noise produced when several channels are present
simultaneously in a cell-attached membrane patch (see Fig.
). Application of heat activates
inward current and current noise in heat-sensitive but not in
heat-insensitive neurons (Fig. A). The power spectrum of
heat-induced noise (Fig. B) indicates the existence of two
Lorentzian components with time constants τ 1 =
17 ms and τ 2 = 0.49 ms. A simple two-state
model reproducing these features is
where τ 1 is the time constant of
transition from the closed state C 1 to
C 2 and τ 2 is the time
constant of transition from the closed state C 2
to the open state (O). The similarity of τ 1 to
the half-time of channel opening after application of a heat jump (Fig.
A) suggests that it is the transition between
C 1 and C 2 that is the main
temperature-dependent event.
Electrophysiologists who work on cultured sensory neurons tend to think
of their cells as a bimodal population, consisting of nociceptive and
nonnociceptive neurons. Whole-animal physiologists who work on
nociceptors in situ know differently; nociceptors come in
many different varieties, with properties such as heat, mechanical, and
chemical sensitivity present to variable extents in different
single-unit recordings. The main division, of course, is between slowly
conducting, unmyelinated nociceptive nerve fibers, which commonly
respond to a wide range of stimuli (polymodal fibers) and more rapidly
conducting myelinated nerve fibers, which frequently respond to a
smaller subset of noxious stimuli, but amongst which heat-sensitive
units are also commonly encountered ( 12, 13). A corresponding division
of nerve-cell bodies is seen, both in dorsal root ganglia and in
cultured preparations, into small-diameter dark neurons and
large-diameter pale neurons ( 14). Heat sensitivity is, perhaps
reassuringly, seen in both cell types in culture ( 10, 15), but there is
a quantitative difference: the smaller cells have a threshold of around
45°C, whereas larger cells form a different population with a
threshold of 51°C ( 15). Interestingly, only the former population
responds to capsaicin, suggesting that there is more than one
heat-sensitive channel at work in this diverse population of
nociceptors (see further discussion below). Thermosensation in C. elegans. The nematode worm
C. elegans is capable of seeking out a preferred temperature
at which to feed, and mutants unable to detect temperature can
therefore be selected by isolating individuals that stray from
preferred-temperature areas. These worms have a mutation either in a
gene, tax-4, that codes for the α-subunit of an ion
channel gated by cyclic nucleotides ( 16) or in a second gene,
tax-2, that codes for a β-subunit ( 17). The fact that
these channels can be gated by cyclic nucleotides suggests (but does
not prove) that the mechanism of thermosensation is the modulation of
the pathway that controls the level of cyclic nucleotides, rather than
the direct action of heat on the ion channel itself. In this respect,
detection of nonnoxious temperatures in C. elegans is
different from mammalian noxious heat sensation. The latter depends
only on expression of a heat-sensitive ion channel, which can be seen
to function in isolated membrane patches and therefore is not gated by
diffusible messengers controlled by intracellular signaling pathways
( 11, 18, 19), whereas thermosensation in C. elegans seems to
depend on cyclic nucleotides as intracellular messengers, and the
thermosensitive element is therefore presumably some stage in the
pathway modulating the level of cyclic nucleotides. There are many
forms of mammalian thermosensation, as outlined in the introduction,
and it is quite possible that signaling pathways are involved in some
of these, even though they do not seem to be directly responsible for
heat sensation in the nociceptors of higher vertebrates. Sensitization of Nociceptors. The process of sensitization (or
hyperalgesia) is familiar to us all: a stimulus strong enough to cause
tissue damage hurts more with time, and even after the stimulus has
been removed, the damaged area is hypersensitive to touch and to
temperature. This phenomenon can be attributed partly to changes in
pain transmission in the spinal cord or at higher levels, but an
important component results from processes occurring at the site of
injury. A large number of molecules released by tissue damage are known
to act as mediators of hyperalgesia. Examples include neuropeptides,
prostaglandins, histamine, platelet-activating factor, and bradykinin
( 9, 20). With so many different factors able to cause hyperalgesia, it is
perhaps no surprise that more than one cellular mechanism is involved.
One recently elucidated mechanism involves activation of protein kinase
A. External inflammatory messengers such as prostaglandins, serotonin,
and adenosine activate adenylate cyclase and consequently increase the
level of cAMP, leading to activation of protein kinase A ( 21, 22). The
principal physiologically important target of protein kinase A seems to
be a recently identified voltage-sensitive Na channel ( 23), which,
unlike the more usual neuronal Na channel, is not blocked by
tetrodotoxin. The effect of phosphorylation of the
tetrodotoxin-resistant Na channel is to lower its threshold, thereby
making it more likely that an action potential will be elicited ( 21,
22). This membrane ionic current is probably not the only one modulated
by cAMP, as actions on a K + current and on a
voltage and cyclic nucleotide-gated conductance have also been
identified ( 24, 25). All of these cAMP-dependent mechanisms, however,
operate in the same direction, in that they sensitize the nociceptive
nerve terminal to any stimulus that is capable of exciting it, because
the effect is to reduce the threshold for action potential firing,
rather than on the specific receptor current induced by the stimulus. A second and more specific mechanism uses activation of protein kinase
C (PKC) to sensitize the response to heat. Fig.
A shows that the
inflammatory mediator bradykinin potently increases the membrane
current activated by a heat pulse ( 6). Bradykinin is known to activate
both phospholipase C and phospholipase A 2,
thereby releasing a number of potential intracellular signaling
molecules. The enhancement of the heat response can be shown to be due
to activation of PKC, however, and not to other possible intracellular
mediators, because it can be mimicked by phorbol esters, which are
specific activators of PKC (Fig. B). Fig. C
shows that activation of PKC increases the current activated by a heat
stimulus and shifts the relation between temperature and membrane
current to lower temperatures. These observations predict that normally
innocuous temperatures, such as body warmth, will therefore become
painful after sensitization, an observation that corresponds well both
with experiments on intact preparations and with our personal
experience that even the warmth of a hand can cause a sensation of pain
when applied to an injured area of the body. Other evidence supporting
the identity of PKC as an intracellular mediator of sensitization
includes the findings that sensitization can be reversed by PKC
inhibitors and can be prolonged by phosphatase inhibitors, which
prevent dephosphorylation after a protein target has been
phosphorylated by PKC ( 6). The possibility that mediators other than
bradykinin may also employ the PKC pathway to induce sensitization has
not yet been investigated and certainly deserves to be.
Desensitization of Nociceptors. When a long pulse of moderate
heat, insufficiently strong to cause cell damage and to release the
extracellular mediators responsible for sensitization, is applied to a
heat-sensitive nociceptor in vivo, gradual adaptation or
desensitization in the firing frequency is observed ( 26). A similar
phenomenon is seen in isolated nociceptors (Fig.
), showing that desensitization,
unlike sensitization, is intrinsic to the nociceptor. Recent
experiments in our lab have shown that desensitization is triggered by
an influx of calcium ions from the external medium through the
heat-sensitive ion channel. In this respect, desensitization of the
heat response resembles the desensitization in response to prolonged
application of capsaicin ( 27), which is triggered by activation of the
calcium-dependent phosphatase calcineurin by an influx of calcium
through the capsaicin-gated channel itself. These observations suggest
that both the heat-activated channel and the capsaicin-gated channel
are desensitized when dephosphorylated by calcineurin, one of many
similarities between the two (see below). The molecular mechanisms of
sensitization of the heat-activated channel (phosphorylation by PKC;
see above) and desensitization (dephosphorylation by calcineurin) may
therefore be simply complementary aspects of the same process, in which
the heat sensitivity of the channel is regulated by phosphorylation
(see Fig. and discussion below).
The Capsaicin Receptor, Vanilloid Receptor Subtype 1 (VR1), and Its
Relation to Heat Sensation. Capsaicin, the active ingredient of
chili peppers, has been known for some time to depolarize nociceptive
nerve terminals by a direct action on an ion channel ( 28). Capsaicin is
not part of the normal environment of most animals. Therefore, it had
always been supposed that the capsaicin receptor was gated
physiologically by an endogenous agonist, just as the
morphine-receptor family is activated physiologically not by morphine
but by endogenous opiates. Capsaicin-responding neurons can
be activated by low pH, and, as pH can drop considerably during
inflammation, hydrogen ions were a plausible candidate for the
physiological agonist activating these nociceptors ( 28). The capsaicin receptor VR1 has recently been cloned by an ingenious
strategy by using imaging of the increase in internal calcium caused by
application of capsaicin to detect expression of capsaicin-receptor
clones ( 18). The expressed receptor is indeed sensitive to low pH, but,
perhaps more interestingly, it responds to heat like the native heat
receptor, as outlined above. The main points of resemblance are as
follows (see refs. 18 and 19). ( i) The current passing
through both channels is zero at room temperature and increases sharply
above about 42°C. ( ii) The capsaicin receptor is a cation
channel with an ionic selectivity similar to that of the native heat
receptor. ( iii) The single-channel conductance and the
current-voltage relation are similar. ( iv) The open time
constant of VR1, 0.9 ms, is similar to the fast open time constant of
the heat-activated channel (0.5 ms; see above). ( v) The
actions of capsaicin and heat are synergistic on both VR1 and the
native heat receptor ( 19, 29). ( vi) VR1 is expressed
exclusively in small neurons of primary sensory ganglia. There may, however, be one crucial point of difference: the ion current
through VR1 is blocked by the capsaicin-channel antagonists capsazepine
and ruthenium red, whether the current is elicited by capsaicin
application or by heat ( 19). However, in cultured nociceptors, the
current induced by capsaicin is blocked by these agonists ( 28), but the
response to heat does not seem to be ( 30). One particularly interesting feature of VR1 is the interaction between
its heat sensitivity and its pH sensitivity. At normal pH, VR1 is
activated only at temperatures above ≈42°C. Low pH acts as a
sensitizing agent, which reduces the threshold for activation by heat
to ≈30°C at pH 6.3 ( 19). The sensitizing effect of pH explains the
observation that capsaicin receptors are activated by low pH; at a
sufficiently low pH, room temperature is adequate to induce channel
openings ( 19). In inflamed or anoxic tissue, the pH can drop to as low
as 6.0, and at this pH, body temperature would be sufficient to
activate VR1. The pain of inflammation and anoxia may therefore be
explained at least partly by a combined effect of low pH and normally
innocuous temperature on VR1. Is VR1 the only heat-detecting mechanism in nociceptors? Probably not,
in view of the observation by Nagy and Rang ( 15) that the two
properties of heat sensitivity and capsaicin sensitivity (and therefore
presumably expression of VR1) are not absolutely colocalized in sensory
neurons, contrary to an earlier report based on a smaller number of
experiments ( 31). A recent study ( 32) reports the cloning of a
vanilloid receptor-like channel (VRL-1) that is not sensitive to
capsaicin but is gated by temperatures above 52°C. Expression of this
channel may explain the responses to higher temperatures observed in
some capsaicin-insensitive neurons ( 15). How Does the Heat Receptor Work? How ligand-gated or
voltage-gated ion channels might work is intuitively fairly obvious, at
least in terms of general principles. Ligand-gated channels operate
like a lock and key; insertion of the key (the ligand) stabilizes the
open state of the channel. Voltage-gated channels possess a charged
gating unit within the membrane field, such that changes in the
membrane potential move this unit and thereby induce a conformational
change that gates the channel open or closed. How small elevations in
temperature might shift the heat-sensitive channel from the closed to
the open state is less intuitively obvious but must depend on the well
known thermodynamic equation
The change in the equilibrium between closed and open states of
the channel, which depends on the Gibbs free energy change (ΔG), can
be markedly temperature-dependent only if there is a large entropy
difference (ΔS) between the two states. Elevations in temperature (T)
must therefore cause the heat-sensitive ion channel to change from an
ordered to a more disordered state, as occurs during melting of ice or
dissolving of a salt in water. It does not seem that any accessory
protein or signaling pathway is needed to gate the channel, because
heat-sensitive ion channels can be seen in cell-free membrane patches
from nociceptors ( 11) and because VR1 functions as a heat receptor when
heterologously expressed in HEK 293 cells or in Xenopus
oocytes ( 18, 19). The temperature-sensitive gating unit is likely,
therefore, to be intrinsic to the heat-sensitive channel protein. The process of sensitization, which shifts the relation between
temperature and channel opening to lower temperatures (see Fig.
C), must act by stabilizing the more disordered,
higher-temperature state of the channel in such a way that lower
temperatures are needed to induce channel opening. How might this
interesting and physiologically important process be operating? One
possibility is that opposite changes in the charge on either side of
the membrane may be important. The work of Tominaga et al.
( 19) has shown that protonation of an external site of VR1 induces
sensitization, and work in our own lab has shown that phosphorylation
of an internal site of the heat-sensitive receptor induces an
apparently identical sensitized state ( 6). If VR1 and the
heat-sensitive receptors are one and the same, as is suggested by most
lines of evidence (see above), then we can put these two observations
together in a simple (and speculative) model of the sensitization
process (Fig. ). In this model,
addition of positive charge to the external face of the membrane or
addition of negative charge to the internal face have equivalent
effects, with both manipulations leading to stabilization of a
disordered state of the protein and consequently to sensitization of
the response to heat.
| | PKC | protein kinase C | | | VR1 | vanilloid receptor
subtype 1 |
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