Two members of the anoctamin family, a recently discovered group of membrane proteins, have unexpectedly been found to play important roles in nociception and pain. Recent papers from the laboratories of Nikita Gamper at the University of Leeds, UK, and Lily Jan at the University of California, San Francisco, US, suggest that at least two family members, Ano1 and Ano3, modulate the properties of nociceptive dorsal root ganglion (DRG) neurons, determining their sensitivity to painful stimuli.
Gamper’s work elucidates the role of membrane-associated protein complexes in nociception, while Jan’s research suggests that Ano3 channels alter DRG neuron sensitivity by directly interacting with and modulating the activity of Slack channels, a poorly understood type of potassium channel. These papers join a 2012 paper by Uhtaek Oh of Seoul National University, South Korea, which shows that Ano1 acts as a heat sensor in nociceptive neurons both in vitro and in vivo. Members of the anoctamin family, at least one of which is a calcium-activated chloride channel (CaCC), may represent new therapeutic targets for pain.
The work from Gamper and colleagues was published online August 27 in Science Signaling, while the research from Jan and coworkers appeared online July 21 in Nature Neuroscience.
Implicating Ano1 in pain
CaCCs open in response to increases in intracellular calcium, allowing chloride ions to flow across the cell membrane. This movement of chloride, which can be inward or outward depending on the cell type, changes the cell’s resting membrane potential and thus alters its electrical excitability. CaCCs have been known to exist since the early 1980s, based on electrophysiological studies of many different cell types, including neurons, smooth muscle cells, and lung epithelial cells (Berg et al., 2012; Duran and Hartzell, 2011). But until the first anoctamin channel was cloned in 2008, the molecules that carried these calcium-activated chloride currents were unknown.
Early expression studies of the first cloned channel, Ano1, established its identity as a CaCC, and the family name “anoctamin” was devised to designate anion-carrying channels (“an”) with a predicted eight (“oct”) transmembrane spans. However, studies since then suggest that while these membrane proteins have closely related sequences, they have diverse functions, and some may not be channels at all. (Many researchers still call them by their original, more generic name, TMEM16 channels.) A flurry of recent studies has provided details on the roles of these channels in physiological processes ranging from the prevention of polyspermy and the stimulation of pulmonary mucus secretion to the enhancement of olfactory signals.
Until quite recently, however, a role for these channels in nociception was unsuspected. Around the same time that the Ano1 cloning and expression studies were first published, Nikita Gamper and his laboratory group, in collaboration with Hailin Zhang’s group at Hebei Medical University in Shijiazhuang, China, set out to fully characterize the molecular events set in motion by the neuroactive peptide bradykinin. Bradykinin is produced at sites of inflammation and is implicated in both acute and chronic pain. It is one of the most potent endogenous pain-causing substances, but the mechanism by which it produces acute pain was unknown. Gamper and colleagues investigated the effects of bradykinin on cultured rat nociceptive DRG neurons by using standard electrophysiological protocols in which ion conditions are varied and ion channel inhibitors are applied to unmask the identities of the channels whose activities, added together, make up the neuron’s overall electrical response (Liu et al., 2010).
In what he calls a “completely accidental” finding, Gamper and his group found in 2010 that CaCCs played a major role in modulating the response of nociceptive DRG neurons to bradykinin. Further experiments revealed details of the acute pain mechanism. Bradykinin activation of the bradykinin receptor 2 (B2R) initiates inositol triphosphate (IP3) signaling, which in turns leads to the release of calcium stored in the endoplasmic reticulum into the cytoplasm. The resulting increase in cytoplasmic calcium activates CaCCs, making neurons more excitable. The increase in cytoplasmic calcium also inhibits M-type potassium channels, whose activity would otherwise have a dampening effect. These two events lead to membrane depolarization, increased excitability, and the rapid firing of increased numbers of action potentials, sending a signal of acute pain to the spinal cord and ultimately to the brain.
Many events can cause cytoplasmic calcium to become elevated, including the activation of voltage-gated calcium channels (VGCCs), which can bring large amounts of calcium in from outside the cell. As he investigated this mechanism, Gamper questioned how Ano1 could react specifically to the release of calcium from internal stores, as would be necessary if Ano1 is to be activated directly by B2R activation. Moreover, the presence of VGCCs might set up a positive feedback loop, whereby voltage changes across the cell membrane would lead to an influx of calcium, activation of CaCCs, further depolarization, and continued activation of the VGCCs, allowing in yet more calcium. A similar mechanism is already known to operate in olfactory neurons, where it amplifies the electrical signal created by certain odorants. But this type of activity is not consistent with the observed behavior of DRG neurons, which are largely quiescent until activated by painful stimuli. In these cells, such a feedback loop would result in continued activation of pain pathways long after the painful stimulus was gone, defeating the temporal and spatial specificity of the nociceptive system. “This is definitely not what happens normally … it would be very bad,” Gamper said.
Gamper and his group went on to investigate how this feedback loop is prevented from occurring in nociceptive neurons. In their latest publication, first author Xin Jin and colleagues provide evidence that Ano1 does not respond to calcium brought in by VGCCs present in DRG neuronal membranes, but instead is selectively activated by calcium released from internal stores. This is accomplished by sequestering the Ano1 channels in specialized membrane microdomains, known as lipid rafts, where they reside in close association with B2Rs, IP3-1 receptors, and the endoplasmic reticulum, where the internal stores of calcium are kept. These Ano1 signaling complexes provide “not 100 percent, but a very high degree of separation from the global calcium increases mediated by VGCCs,” preserving the specificity of the bradykinin signaling system, Gamper said. This mechanism also helps overcome the very low affinity that Ano1 channels have for calcium by keeping them in close proximity to the calcium source (see comment below on the work from Gamper’s group).
This research is complemented by earlier work from the laboratory of Uhtaek Oh, who reported in 2012 that, in addition to being activated by internal calcium, Ano1 is activated by noxious heat. Oh and his group investigated the activity of Ano1 channels by studying their activity in cultured mouse DRG neurons and by exogenously expressing Ano1 in HEK293 cells, a frequently used system for analyzing the activities of cloned channels. The discovery of Ano1’s heat sensitivity adds to nociceptive neurons’ repertoire of molecules that detect noxious temperatures, which also includes multiple members of the transient receptor potential (TRP) channel family as well as STIM1, an endoplasmic reticulum protein that interacts with the Orai1 calcium release-activated calcium channel to produce a calcium signal when temperatures are elevated.
Although it seems redundant, "thermoception is important in normal life. It would be risky if heat sensing is not redundant and the heat sensor is out of order,” Oh said. Oh’s work using thermal pain models in mice also implicated Ano1 channels in the augmentation of chronic pain, and he is preparing a manuscript dealing with this aspect of Ano1’s function. Oh’s studies suggest that inhibition of Ano1 could be a therapeutic target for pain.
In his paper, Oh noted an interesting paradox. Because Ano1 is a chloride channel, activation of Ano1 is under consideration as a therapeutic mechanism for the treatment of cystic fibrosis, an inherited disorder that results from defects in the chloride channel CFTR. However, Ano1’s role as a sensor for nociceptive stimuli might mean that such treatments would have painful side effects.
A role for Ano3 via Slack
Ano3 is a somewhat more mysterious member of the anoctamin family, but one whose importance is highlighted by its wide expression in brain tissues, including the human striatum, hippocampus, and cortex. Exome sequencing studies have linked an Ano3 mutation to human autosomal-dominant craniocervical dystonia, a movement disorder affecting the muscles of the head and neck. Lily Jan and her group investigated the properties of Ano3 knockout rats, which show increased sensitivity to painful thermal and mechanical stimuli. While investigating the electrophysiological properties of nociceptive DRG neurons from these animals, they made the unexpected discovery that lack of Ano3 expression greatly reduced expression of the sodium-activated potassium channel Slack.
Slack has been studied in multiple settings (Salkoff et al., 2006) and is thought to modulate neuronal firing patterns by opening in response to changes in intracellular sodium concentrations brought about by action potentials. However, only a few studies (Bischoff et al., 1998; Tamsett et al., 2009; Nuwer et al., 2010) had previously linked it to nociception. Jan’s group investigated potential interactions between Ano3 and Slack by expressing the proteins in HEK293 cells, separately and together. First author Fen Huang and colleagues could not detect current generated by Ano3 itself in these cells. Instead, their experiments showed that Ano3 physically interacts with Slack to increase its activity by several mechanisms: increasing the probability of full opening of single channels at low sodium concentrations, increasing its sodium sensitivity, and stabilizing its expression in the cell membrane. In nociceptive DRG neurons, Slack has a dampening effect on excitability, and Ano3 would enhance that effect by promoting Slack activity. Thus, a lack of Ano3 led to nociceptive hypersensitivity in the rat knockouts (see comments below on the work from Jan’s group and commentary in Nature Neuroscience by Gadotti and Zamponi, 2013).
“The interactions between the proteins could dial pain sensations up or down,” Jan said, thus conferring great sensitivity and selectivity on the pain signaling system. Together with the findings of Oh and Gamper, these observations suggest that modulation of pain is a key function for at least some anoctamin family members.
Jan cautioned that her work does not rule out the possibility that Ano3 has channel activity of its own when expressed in other cell types. She and Gamper both noted that experiments conducted in exogenous cell systems or even isolated neurons may not exactly reflect what is happening in the whole animal. In addition, most studies are carried out using electrophysiological recordings from cell bodies, because the nociceptive neuronal endings, where pain sensations are initiated, are small, heterogeneous, and challenging to work with. In animals and humans, nociceptive DRG neuronal cell bodies are located near the spinal cord, far from the action at the periphery.
Gamper says that he and his group are “intrigued” by the B2R- and Ano1 channel-carrying membrane microdomains, and plan to continue examining their structure and function. Jan’s group intends to continue study of Ano3 in nociceptive neurons as well as in the brain, perhaps with the help of conditional knockouts that will allow them to selectively remove the protein from nociceptive neuron endings or specific brain regions. And, as mentioned above, Oh is pursuing studies on the role of Ano1 in mediating chronic pain. Much remains to be learned from these intriguing new players in pain.
Megan M. Stephan, PhD, is a science and medical writer based in Cambridge, Massachusetts, US.
Comments on Related Content
Arin Bhattacharjee, The State University of New York at Buffalo
In neuropathic pain states,
In neuropathic pain states, there are changes in ion conductances within sensory neurons, resulting in hyperexcitability. However, identification of the principal ion channels responsible for the observed hyperexcitability in neuropathic pain has been elusive. The Lily Jan lab has just published a paper that implicates Na+-activated Slack potassium channels as important for pain signaling, and this may mean that the altered regulation of these channels underlie neuropathic pain.
In the paper, Huang et al. document that the TMEM16C subunit (aka Ano3) may regulate the expression and Na+-dependent properties of Slack. Using a gene trap method, they successfully knocked down TMEM16C in rats and found that this resulted in diminished Slack expression, broadening of DRG neuronal action potentials, thermal hyperalgesia, and mechanical allodynia. When expressed alone in heterologous expression systems, TMEM16C, a member of the Ca2+-activated Cl- channel family, did not produce any currents, but when co-expressed with Slack channels are able to enhance the Na+-dependence of Slack channels. Our group had previously shown that knocking down Slack channels by siRNA in cultured DRG neurons resulted in depolarization and a loss of firing accommodation (Nuwer et al., 2010). Huang et al. extend our initial findings by now demonstrating that intrathecal injections of siRNAs specific to Slack resulted in neuropathic pain-like behavior: thermal hyperalgesia and mechanical allodynia.
There are some discrepancies with their findings to what we have observed. First, it seems that TMEM16C is only expressed in IB+, non-peptidergic neurons. We and others have reported Slack KNa channels expressed in all DRG neurons. So why TMEM16C only modulates these channels in IB+ cells is not clear. In HEK cells, TMEM16C shifted the Na+ dependence of Slack channels from an EC50 of 75 mM to 45 mM and seems to lower the unitary conductance of Slack channels to ~110 pS. Our group has shown, recording from DRG neurons, the EC50 for Na+ to be 50 mM, which can then be shifted to a physiological range of 20 mM by NAD+ (Tamsett et al., 2009). We reported the unitary conductance of native KNa channels to be 200 pS. It is unfortunate that Huang et al. did not test Na+ dependence and unitary conductance of KNa channels in DRG neurons from TMEM16 knockout rats. I bring these points up because they are important in understanding why Slack channels are expressed in DRG neurons. Slack channels have intrinsic voltage dependence and, in fact, behave like delayed rectifiers (Budelli et al., 2009). For DRG neurons, as long as Slack can operate in the physiological range of intracellular Na+, it is energetically cheaper to express Slack channels compared to typical voltage-dependent potassium channels, which usually have conductances ranging from ~10-20 pS. You need far fewer Slack channels to generate an appreciable K+ conductance compared to run-of-the-mill voltage-dependent potassium channels, and for neurons whose fibers can be very, very long, this may be important. Nonetheless, the findings by Huang et al. have put the spotlight on these channels with respect to pain. It remains to be seen if the downregulation of Slack channels are indeed responsible for neuropathic pain. Meta-analysis of published gene expression studies have shown that the Slack gene is downregulated in the spinal cord injury of neuropathic pain (Courade et al., 2011).
Uhtaek Oh, Seoul National University
This paper elegantly
This paper elegantly describes the role of ANO3/TMEM16C in the pain sensory system. ANO1 and ANO2 are Cl- channels activated by intracellular Ca2+. So, the authors seemed to expect that ANO3 is a Cl- channel that would regulate pain transmission, because ANO3 is expressed in DRG neurons, largely IB4-positive nociceptors. However, they failed to find that ANO3 is a Cl- channel activated by Ca2+. Instead, the authors found that ANO3 upregulates the activity of Slack, a Na+-activated K+ channel. Using ANO3-deficient rats and other biochemical techniques, the authors elegantly showed that ANO3 physically binds Slack and augments Slack channel activity. Therefore, when ANO3 is deficient, Slack channels are less active in vivo, which leads to augmented excitability of nociceptors, lowering the pain threshold. Thus, the present study provides two important findings. 1) The paper suggests that Slack channels serve as a brake on pain transmission. 2) ANO3/TMEM16C also suppresses pain transmission in association with Slack.
Allan Basbaum, University of California
This comment was co-authored
This comment was co-authored by Fen Huang and Lily Jan, both at University of California San Francisco, US.
We appreciate the thoughtful comments on our paper, and are happy to discuss the points raised by Dr. Bhattacharjee who has published several very relevant studies on Slack channels in DRG neurons.
The first point relates to our finding that TMEM16C is primarily expressed in IB4+, non-peptidergic neurons. Because Dr. Bhattacharjee and others have reported that Slack KNa channels are expressed in all DRG neurons, it was unexpected that TMEM16C only modulates these channels in IB4+ cells. We prefer to avoid what may be a theological question as to why TMEM16C is predominantly expressed in IB4+ cells. While it is interesting to contemplate questions like this one, we cannot provide an answer.
It may be significant that we used different antibodies for Slack immunostaining of DRG neurons (chicken polyclonal antibody against N-terminal peptide of Slack in Tamsett et al., 2009, compared to mouse monoclonal antibody from Neuromab against C-terminal peptide of Slack in our study). This may account for the difference in staining pattern, in which we find Slack expression predominantly in IB4+ cells. Future studies with genetically modified animal models or specific pharmacological blockers for Slack channels will be necessary to establish the definitive expression pattern of Slack and its precise contribution to pain processing.
As to the sodium sensitivity, our measurement of EC50 in HEK293 cells co-expressing TMEM16C and Slack actually matches very well with the reported EC50 in native DRG neurons (~50 mM; Tamsett et al., 2009). NAD modulation could further increase the sodium sensitivity to 20 mM (Tamsett et al., 2009). Indeed modulation of EC50 by NAD, as well as by associated proteins, such as TMEM16C, could allow KNa channels to operate under physiologically relevant levels of intracellular Na+, which could explain how KNa channels can control normal neuronal excitability.
Literature reports of single channel conductance of KNa channels vary significantly. It appears that the channel conductance is dependent on cell types and on recording conditions, particularly the composition of the intracellular and extracellular solutions. For example, the Slack channel single channel conductance in oocytes is around 88 pS with 80 mM K+o/ K+i (80 mM Na+, 160 mM Cl-), while with 160 mM K+o/K+i, the single channel conductance is around 165 pS (Yuan et al., 2003). Joiner and colleagues (Joiner et al., 1998) stated that “this intrinsic rectification was somewhat smaller in symmetric K+: the unitary chord conductance of Slack channels measured between -40 mV and +60 mV (from a calculated EK of 0 mV) varied from 40 to 65 pS, respectively” and “the mean current levels at +20 mV, determined from amplitude histograms, were multiples of the unitary current and corresponded to a single channel conductance of 43 pS”. It has also been reported to have a unitary conductance of approximately 60 pS (Bhattacharjee et al., 2002). In another paper, Santi and colleagues (Santi et al., 2006) stated in the figure legend of Figure 4 that “the unitary conductance was similar to Slo2.2 (88 pS).”
The native KNa channels in DRG neurons reported to have a single channel conductance of ~200 pS were recorded with symmetric solutions with 10 mM Na+, 130 mM K+ and 1 mM TEA (Tamsett et al., 2009). It is, of course, possible that TMEM16C contributes to the native KNa conductance in DRG neurons. Furthermore, we do not know the precise molecular components of the KNa channels in native DRG neurons. In another case, the native KNa channels in MNTB neurons exhibit a large-conductance of 122 pS and a smaller- conductance (<40 pS) when recorded with 130 mM K+i / 2.5 mM K+o, 10 mM Na+i, 34.5 mM Cl-i (Yang et al., 2007).
In our recordings, we used two different sets of intracellular and extracellular solutions in the inside-out excised patch experiments. We used an all point histogram and Gaussian fit to obtain the major unitary current amplitude at each voltage and, as shown in Figure 6c, the relationships between current amplitude and voltage in various recording solutions can be adequately fit with linear functions. In cells expressing TMEM16C and Slack, the full single channel conductance is ~110 pS with 140 mM [K+]o /140 mM [K+]i, 0 mM [Na+]i, and the mean single channel conductance is around 80 pS with 140 mM [K+]o / 60 mM [K+]i, 0 mM [Na+]i.
We agree that it will be interesting to determine the Na+ sensitivity and unitary conductance of KNa channels in DRG neurons from TMEM16 knockout rats. Unfortunately, our physiological rig setting for single channel recording does not allow us to visualize the live cell labeling of IB4-FITC in the cultured DRG neurons. The reduction of Slack protein levels in the TMEM16C KO neurons presents further challenges for reliably detecting KNa single channel activities and characterization of their properties. Unquestionably, it will be of interest to pursue these questions in future studies.
Arin Bhattacharjee, The State University of New York at Buffalo
I would like to thank Dr. Basbaum for responding to my comments on their paper. I would also like to take this opportunity to respectfully respond to some of the points Dr. Basbaum raised in his commentary.
First, in our paper, in addition to the immunohistochemical analyses of Slack (Tamsett et al., 2009) within rat DRG, we also provided electrophysiological evidence that KNa channels can be recorded from both small and large DRG neurons. Second, in-situ analyses of Slack gene expression taken from the spinal cords of four-week-old mice by the Allen institute also indicate that Slack is ubiquitously expressed in the DRG, corroborating with our data. So I believe that the restricted Slack expression in DRG neurons as reported by Huang et al. remains an unsettled issue.
Regarding the unitary conductance, I personally recorded Slack channels from transfected CHO cells and found the unitary conductance to be ~180 pS in symmetrical K (130/130) (Bhattacharjee et al., 2003). This is consistent with what we recorded in DRG neurons. The 88 pS recorded by the Salkoff group (Santi et al., 2006) can be explained by the fact that they used 80 mM symmetrical K+ instead of 130 mM. Lowering the K+ concentration will lower the unitary conductance. In the original Slack cloning paper (Joiner et al., 1998), single channel recordings were made under Na+-free conditions. The Kazcmarek group repeated Slack expression studies in Xenopus oocytes (Chen et al., 2009), and when they recorded Slack channels in a symmetrical 140K, they also reported a unitary conductance of 180 pS. So no matter how you examine it, the Slack unitary conductance recorded in various heterologous expression systems by various groups differs considerably from the smaller -110 pS reported by Huang et al. I assumed that it was TMEM16C associating with Slack that was lowering the unitary conductance. Unfortunately, the unitary conductance for Slack with and without TMEM16C in Na+-containing solutions was not reported. I still believe it is imperative to study residual KNa channels in TMEM16C knockout rats, and I hope the authors will consider doing so in the future.
Frédérique Scamps, INSERM
The study by Jin and
The study by Jin and coworkers decrypts the cellular and molecular basis accounting for the specific receptor-dependent activation of the calcium-activated chloride current, CaCC, expressed in peripheral nociceptive neurons. CaCCs are widely expressed in numerous cells including neurons and, by definition, they require an increase in intracellular calcium concentration for opening. To understand their role, the source of calcium responsible for their activation has systematically been investigated. Until recently, no specificity was observed regarding Ca2+ origin: Ca2+ entry through voltage-gated calcium current (VGCC) or receptor-mediated intracellular Ca2+ mobilization were both effective for activating neuronal CaCCs. In their initial study (Liu et al., 2010), Nikita Gamper’s group revealed that CaCC could be specifically activated by receptor-mediated Ca2+ increase. Bradykinin-induced intracellular Ca2+ release from the endoplasmic reticulum, but not VGCC, activated a CaCC in small nociceptive sensory neurons. The follow-up of this important study is now addressed by Jin and coworkers, who deciphered why VGCC was unable to activate CaCC in those neurons. Their working hypothesis is based on the low Ca2+ sensitivity of Ano1, the channel mediating the CaCC recorded in nociceptors, which implies that Ano1 must face micromoles of Ca2+ for opening. They demonstrate that lipid rafts, which are membrane caveolae, are scaffolds for Ano1 and plasma membrane receptors. In addition, interaction between Ano1 and IP3R allows sufficient increase in intracellular Ca2+ to activate the poorly Ca2+-sensitive Ano1-CaCC
Remarkably, the physical interactions identified by Jin and colleagues could account for local signaling responses and functional specificity. So, unlike Ano2-CaCC expressed in hippocampal neurons (Huang et al., 2012), this study establishes that Ano1 cannot be involved in VGCC-dependent synaptic activity. It has been proposed that a putative CaCC-mediated depolarization could be an important element of the generator potential in nociceptive terminals (Granados-Soto et al., 2005), a hypothesis in agreement with the current study. To further support such a role for CaCC in nociception, it would be of interest to determine the subcellular localization of Ano1 in the peripheral pain circuitry.