Citation: Fortune ES, Chacron MJ (2009) From Molecules to Behavior: Organismal-Level Regulation of Ion Channel Trafficking. PLoS Biol 7(9): e1000211. https://doi.org/10.1371/journal.pbio.1000211
Published: September 29, 2009
Copyright: © 2009 . This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by a National Science Foundation grant (IOB-0543985; ESF) and funding from the Canadian Institutes of Health Research, Canada Foundation for Innovation, and Canada Research Chairs (MJC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Abbreviations: EOD, electric organ discharge
Transmembrane proteins are critical, not only for cell survival, but also for a myriad of physiological functions in multicellular organisms. It is therefore necessary to have mechanisms that regulate the number of these proteins present on the cellular membrane at any given time. One such mechanism involves protein trafficking, or constitutive cycling, in which transmembrane proteins are continuously transferred between a pool located within the endoplasmic reticulum and the membrane surface by shuttle proteins (Figure 1) . The actual number of proteins at the membrane surface is controlled by the rate of exocytosis (i.e., the rate at which proteins are inserted into the membrane) as well as the rate of endocytosis (i.e., the rate at which proteins are removed from the membrane). A higher rate of exocytosis will increase the number of proteins at the membrane surface, whereas a lower rate of exocytosis will decrease that number –.
Ion channels from a pool within the endoplasmic reticulum are moved to the membrane surface via shuttle proteins, a process called exocytosis. Ion channels can also be moved from the membrane surface back to the endoplasmic reticulum via different shuttle proteins, a process called endocytosis. Both endocytosis and exocytosis occur continuously, and one thus characterizes them by their rates, which are simply the number of transmembrane proteins being removed and inserted from the membrane per unit time, respectively. Both rates can be independently regulated.
Receptor Trafficking as a Mechanism for the Regulation of Transmembrane Proteins
Both exocytosis and endocytosis of transmembrane proteins involve distinct agents, including chaperones, glycosylases, microtubule systems, actin, as well as myosin , and can be independently regulated by several known mechanisms –, including circulating hormones ,. Ever since its proposal in the 1970s in the context of gastric acid regulation ,, protein trafficking has been shown to have a crucial role in a variety of physiological functions, including the regulation of gastric acid ,, osmolarity through water transport , glucose levels , and regulation of various ionic concentrations –.
In particular, protein trafficking has also been shown to occur in excitable cells such as cardiac myocytes , and neurons ,. The hippocampus has received perhaps the most attention in this regard; trafficking has been demonstrated to have a critical role in determining the synaptic dynamics involved in synaptic plasticity, which is thought to underlie learning and memory. Increases and decreases in AMPA receptor trafficking are correlated with long-term potentiation and depression of synapses, respectively. Further, ion channels can be inserted in and out of the neuronal membrane in a continuous fashion along with receptors during plasticity ,.
As the molecular basis for channel and receptor trafficking are studied, we need to be cognizant of the cellular and organismal consequences of these mechanisms. Continuous channel and receptor trafficking appears to be a ubiquitous mechanism in both vertebrate and invertebrate animals. At first glance, this sort of trafficking of transmembrane proteins appears to be metabolically costly. What benefits, if any, do these trafficking mechanisms afford physiological systems over other mechanisms such as protein synthesis and degradation? Answering this important question will require an integrative approach that relates the molecular basis of trafficking to changes in cell function and behavior. Specialized animal model systems have historically proven to be useful in such multilevel integrative studies –.
Weakly Electric Fishes as a Model System for the Study of Receptor Trafficking
Weakly electric fish have a suite of simple physiological and behavioral adaptations that make them ideal for studying the physiological basis and evolution of behavior. These adaptations relate to these fishes' ability to generate an electric field (the electric organ discharge, or EOD) around their body, which is detected by electroreceptors in the skin (Figure 2) . This active electric sense is used in a wide variety of evolutionary and ecologically important functions, including prey location and capture  and communication with conspecifics ,.
(A) Electrogenesis in Sternopygus macrurus. Adult fish are on the order of tens of centimeters in length. The electric organ is located along the tail and produces a quasisinusoidal electric field whose frequency varies between 40 and 200 Hz. The electric organ, which is roughly located where the white stripe on the side of the fish appears, is composed of electrocytes with voltage-gated sodium and potassium channels that are concentrated on the caudal aspect of the cell membrane, arranged in series (inset). The currents generated by these channels summed over all electrocytes give rise to a potential difference between the inside and outside of the fish that propagates through the water at the speed of light. The colors surrounding the photograph of the fish correspond to the relative strength of the electric field. In this snapshot of the electric field, the region around the body and head is positive (blue colors) and the region around the tail is negative (red colors). At other times, the positive and negative areas are reversed. This field is detected by electroreceptors that are embedded in the skin. (B) Sternopygus at night or after social interactions. These conditions lead to an increase in the circulating ACTH, which increases the rate of exocytosis of channels in the electrocytes, thereby increasing their density (inset). This, in turn, increases the intensity of the electric field and, therefore, the distance at which the electric field propagates in the water. As a result, salient objects like prey items (purple dot) may be detected at greater distances. (C) Sternopygus during the day or in solitary conditions. Under these circumstances, there is a smaller rate of exocytosis due to lower levels of ACTH and thus fewer channels in the electrocytes (inset). As a result, the fish produces a weaker electric field that will decay over smaller distances. Because electrosensory perception is dependent on the detection of voltage differences in the water, this reduced electric field is less effective for detecting prey and for communicating to nearby conspecifics. Sternopygus photograph courtesy of Scott Shulz.
The EOD results from the sum of ionic currents produced by specialized excitable cells in the electric organ called electrocytes, which are modified muscle cells . In Sternopygus macrurus, a species of South American gymnotiform weakly electric fish commonly known as longtail or goldline knifefish, a detailed description of the ionic and molecular basis for the generation of the sinusoidal electric field has been achieved . The electrocytes use a combination of specialized excitatory sodium channels and potassium channels to generate one action potential per EOD cycle: the kinetics and relative distribution of these channels are the sole determinants of EOD magnitude and duration ,. The EOD duration is used to communicate the sex and social status of an individual fish , whereas the EOD amplitude will effectively determine the animal's sensing volume (i.e., the volume around its body within which it can detect objects such as prey or conspecifics) as well as the emission volume of communication signals (Figure 2) . Previous studies have shown that various hormones such as androgens and estrogens will modulate EOD duration through the regulation of both sodium and potassium conductances in electrocytes . This level of detail between ion channel regulation in excitable cells and an easily measured behavioral output has not been described in other vertebrate systems.
A New Functional Role for Receptor Trafficking and a New Mechanism for Its Regulation
In this issue of PLoS Biology, Markham et al.  found that social and circadian environmental factors result in dramatic changes in the amplitude of the electric field of individual Sternopygus: specifically, they found that Sternopygus increases its EOD amplitude at nighttime, when the animal is most active, hunting for prey and interacting with conspecifics.
The authors then examine the hierarchy of mechanisms that underlie this organismal-level phenomenon. Because circadian variations in the pituitary adrenocorticotropic hormone (ACTH) have been observed in a variety of vertebrate species , Markham et al. hypothesized that the circadian variations in the EOD amplitude of Sternopygus were due to changing levels of ACTH as was observed for another species of weakly electric fish, Brachyhypopomus pinnicaudatus . The authors found that injection of exogenous ACTH into the animal leads to increases in the EOD amplitude during the day. This phenomenon can be reproduced in isolated electrocytes from these animals: the application of ACTH causes an increase in the amplitude of the action potential, which directly determines EOD amplitude.
What are the mechanisms that underlie increased action potential height? One hypothesis is that this phenomenon is due to an increased number of sodium channels at the membrane surface, which could be due to a higher rate of channel exocytosis. Markham et al.  show that ACTH affects a cAMP/PKA pathway to up-regulate two distinct ionic currents, a Na+ current and an inward rectifier K+ current, by increasing exocytosis of the two transmembrane molecules that mediate these currents. A delayed rectifier K+ current that is also found in these cells is not regulated by this mechanism. Thus, social cues lead to increased circulating ACTH, which modulates intracellular cAMP/PKA, which in turn increases the rate of sodium channel insertion into the membrane of the electrocyte. This increase in sodium channels leads to an increase in EOD amplitude, which will improve the distance at which detection of behaviorally relevant stimuli will occur .
Organismal Approaches to Understanding Channel and Receptor Trafficking
The results of Markham et al. are an example of how an organismal perspective can be used to elucidate the functional roles of subcellular phenomena in evolutionarily relevant behaviors. This work has shown new modes for the regulation of ion channel trafficking, including circadian and social cues. This has important implications for the study of protein trafficking in general as environmental factors can now be used as an additional tool to study this phenomenon in other systems. Of particular interest are cardiac myocytes, which display many similarities with electrocytes , and sodium channel trafficking ,, which can also be regulated by hormones .
Interestingly, Markham et al.  show that only two of the three ion channels present in electrocytes are up-regulated by ACTH, raising an important question regarding specificity: What makes a particular transmembrane protein a target for up-regulation or down-regulation? Furthermore, can different transmembrane proteins be trafficked by the same shuttle protein? Further studies are needed to address these issues.
The work of Markham et al.  also begins to address the important question of identifying the putative advantages of having constitutive cycling of transmembrane proteins, which is metabolically costly to the organism . One possible advantage is that constitutive cycling permits responsiveness to circulating levels of hormones on a relatively short timescale that does not need protein synthesis –. We can expect that many animal systems have behaviors in which hormone titers can be expected to regulate cell excitability on a relatively fast timescale. For example, social conditions and song production are known to modulate circulating levels of hormones in songbirds . These hormones, which can be regulated by the animal's behavior, in turn affect animal behavior, forming a feedback circuit from brain mechanisms through behavior. In this context, studies that examine the interplay between molecular mechanisms and behavior using the same sort of organismal approach that was used by Markham and colleagues are likely to make significant progress towards this goal.
- 1. Royle S. J, Murrell-Lagnado R. D (2003) Constitutive cycling: a general mechanism to regulate cell surface proteins. Bioessays 25: 39–46.S. J. RoyleR. D. Murrell-Lagnado2003Constitutive cycling: a general mechanism to regulate cell surface proteins.Bioessays253946
- 2. Zhou J, Yi J, Hu N, George A. L Jr, Murray K. T (2000) Activation of protein kinase A modulates trafficking of the human cardiac sodium channel in Xenopus oocytes. Circ Res 87: 33–38.J. ZhouJ. YiN. HuA. L. George JrK. T. Murray2000Activation of protein kinase A modulates trafficking of the human cardiac sodium channel in Xenopus oocytes.Circ Res873338
- 3. Cusdin F. S, Clare J. J, Jackson A. P (2008) Trafficking and cellular distribution of voltage-gated sodium channels. Traffic 9: 17–26.F. S. CusdinJ. J. ClareA. P. Jackson2008Trafficking and cellular distribution of voltage-gated sodium channels.Traffic91726
- 4. Abriel H (2007) Roles and regulation of the cardiac sodium channel Na v 1.5: recent insights from experimental studies. Cardiovasc Res 76: 381–389.H. Abriel2007Roles and regulation of the cardiac sodium channel Na v 1.5: recent insights from experimental studies.Cardiovasc Res76381389
- 5. Reuter H, Scholz H (1977) The regulation of the calcium conductance of cardiac muscle by adrenaline. J Physiol 264: 49–62.H. ReuterH. Scholz1977The regulation of the calcium conductance of cardiac muscle by adrenaline.J Physiol2644962
- 6. Forte T. M, Machen T. E, Forte J. G (1977) Ultrastructural changes in oxyntic cells associated with secretory function: a membrane-recycling hypothesis. Gastroenterology 73: 941–955.T. M. ForteT. E. MachenJ. G. Forte1977Ultrastructural changes in oxyntic cells associated with secretory function: a membrane-recycling hypothesis.Gastroenterology73941955
- 7. Okamoto C. T, Forte J. G (2001) Vesicular trafficking machinery, the actin cytoskeleton, and H+-K+-ATPase recycling in the gastric parietal cell. J Physiol 532: 287–296.C. T. OkamotoJ. G. Forte2001Vesicular trafficking machinery, the actin cytoskeleton, and H+-K+-ATPase recycling in the gastric parietal cell.J Physiol532287296
- 8. Nielsen S, Frokiaer J, Marples D, Kwon T. H, Agre P, et al. (2002) Aquaporins in the kidney: from molecules to medicine. Physiol Rev 82: 205–244.S. NielsenJ. FrokiaerD. MarplesT. H. KwonP. Agre2002Aquaporins in the kidney: from molecules to medicine.Physiol Rev82205244
- 9. Pessin J. E, Thurmond D. C, Elmendorf J. S, Coker K. J, Okada S (1999) Molecular basis of insulin-stimulated GLUT4 vesicle trafficking. Location! Location! Location! J Biol Chem 274: 2593–2596.J. E. PessinD. C. ThurmondJ. S. ElmendorfK. J. CokerS. Okada1999Molecular basis of insulin-stimulated GLUT4 vesicle trafficking. Location! Location! Location!J Biol Chem27425932596
- 10. Kanzaki M, Zhang Y. Q, Mashima H, Li L, Shibata H, et al. (1999) Translocation of a calcium-permeable cation channel induced by insulin-like growth factor-I. Nat Cell Biol 1: 165–170.M. KanzakiY. Q. ZhangH. MashimaL. LiH. Shibata1999Translocation of a calcium-permeable cation channel induced by insulin-like growth factor-I.Nat Cell Biol1165170
- 11. Bobanovic L. K, Royle S. J, Murrell-Lagnado R. D (2002) P2X receptor trafficking in neurons is subunit specific. J Neurosci 22: 4814–4824.L. K. BobanovicS. J. RoyleR. D. Murrell-Lagnado2002P2X receptor trafficking in neurons is subunit specific.J Neurosci2248144824
- 12. Rotin D, Kanelis V, Schild L (2001) Trafficking and cell surface stability of ENaC. Am J Physiol Renal Physiol 281: F391–399.D. RotinV. KanelisL. Schild2001Trafficking and cell surface stability of ENaC.Am J Physiol Renal Physiol281F391399
- 13. Malinow R, Malenka R. C (2002) AMPA receptor trafficking and synaptic plasticity. Ann Rev Neurosci 25: 103–126.R. MalinowR. C. Malenka2002AMPA receptor trafficking and synaptic plasticity.Ann Rev Neurosci25103126
- 14. Lai H. C, Jan L. Y (2006) The distribution and targeting of neuronal voltage-gated ion channels. Nat Rev Neurosci 7: 548–562.H. C. LaiL. Y. Jan2006The distribution and targeting of neuronal voltage-gated ion channels.Nat Rev Neurosci7548562
- 15. Faber E. S, Delaney A. J, Power J. M, Sedlak P. L, Crane J. W, et al. (2008) Modulation of SK channel trafficking by beta adrenoceptors enhances excitatory synaptic transmission and plasticity in the amygdala. J Neurosci 28: 10803–10813.E. S. FaberA. J. DelaneyJ. M. PowerP. L. SedlakJ. W. Crane2008Modulation of SK channel trafficking by beta adrenoceptors enhances excitatory synaptic transmission and plasticity in the amygdala.J Neurosci281080310813
- 16. Kim J, Jung S. C, Clemens A. M, Petralia R. S, Hoffman D. A (2007) Regulation of dendritic excitability by activity-dependent trafficking of the A-type K+ channel subunit Kv4.2 in hippocampal neurons. Neuron 54: 933–947.J. KimS. C. JungA. M. ClemensR. S. PetraliaD. A. Hoffman2007Regulation of dendritic excitability by activity-dependent trafficking of the A-type K+ channel subunit Kv4.2 in hippocampal neurons.Neuron54933947
- 17. Kandel E. R (1997) Genes, synapses, and long-term memory. J Cell Physiol 173: 124–125.E. R. Kandel1997Genes, synapses, and long-term memory.J Cell Physiol173124125
- 18. Coleman B. D, Renninger G. H (1974) Theory of delayed lateral inhibition in the compound eye of limulus. Proc Natl Acad Sci U S A 71: 2887–2891.B. D. ColemanG. H. Renninger1974Theory of delayed lateral inhibition in the compound eye of limulus.Proc Natl Acad Sci U S A7128872891
- 19. Heiligenberg W (1991) Neural nets in electric fish. Cambridge, MA: MIT Press. 179 p.W. Heiligenberg1991Neural nets in electric fishCambridge, MAMIT Press179
- 20. Turner R. W, Maler L, Burrows M (1999) Electroreception and electrocommunication. J Exp Biol 202: 1167–1458.R. W. TurnerL. MalerM. Burrows1999Electroreception and electrocommunication.J Exp Biol20211671458
- 21. Zakon H. H, Oestreich J, Tallarovic S, Triefenbach F (2002) EOD modulations of brown ghost electric fish: JARs, chirps, rises, and dips. J Physiol (Paris) 96: 451–458.H. H. ZakonJ. OestreichS. TallarovicF. Triefenbach2002EOD modulations of brown ghost electric fish: JARs, chirps, rises, and dips.J Physiol (Paris)96451458
- 22. Zakon H. H, Dunlap K. D (1999) Sex steroids and communication signals in electric fish: a tale of two species. Brain Behav Evol 54: 61–69.H. H. ZakonK. D. Dunlap1999Sex steroids and communication signals in electric fish: a tale of two species.Brain Behav Evol546169
- 23. Unguez G. A, Zakon H. H (1998) Phenotypic conversion of distinct muscle fiber populations to electrocytes in a weakly electric fish. J Comp Neurol 399: 20–34.G. A. UnguezH. H. Zakon1998Phenotypic conversion of distinct muscle fiber populations to electrocytes in a weakly electric fish.J Comp Neurol3992034
- 24. Zakon H, McAnelly L, Smith G. T, Dunlap K, Lopreato G, et al. (1999) Plasticity of the electric organ discharge: implications for the regulation of ionic currents. J Exp Biol 202: 1409–1416.H. ZakonL. McAnellyG. T. SmithK. DunlapG. Lopreato1999Plasticity of the electric organ discharge: implications for the regulation of ionic currents.J Exp Biol20214091416
- 25. Ferrari M. B, Zakon H. H (1993) Conductances contributing to the action potential of Sternopygus electrocytes. J Comp Physiol A 173: 281–292.M. B. FerrariH. H. Zakon1993Conductances contributing to the action potential of Sternopygus electrocytes.J Comp Physiol A173281292
- 26. Mills A, Zakon H. H (1991) Chronic androgen treatment increases action potential duration in the electric organ of Sternopygus. J Neurosci 11: 2349–2361.A. MillsH. H. Zakon1991Chronic androgen treatment increases action potential duration in the electric organ of Sternopygus.J Neurosci1123492361
- 27. Schaefer J, Zakon H. H (1996) Opposing actions of androgen and estrogen on in vitro firing frequency of neuronal oscillators in the electromotor system. J Neurosci 16: 2860–2868.J. SchaeferH. H. Zakon1996Opposing actions of androgen and estrogen on in vitro firing frequency of neuronal oscillators in the electromotor system.J Neurosci1628602868
- 28. Snyder J. B, Nelson M. E, Burdick J. W, Maciver M. A (2007) Omnidirectional sensory and motor volumes in electric fish. PLoS Biol 5: e301.J. B. SnyderM. E. NelsonJ. W. BurdickM. A. Maciver2007Omnidirectional sensory and motor volumes in electric fish.PLoS Biol5e301
- 29. Stoddard P. K, Zakon H. H, Markham M. R, McAnelly L (2006) Regulation and modulation of electric waveforms in gymnotiform electric fish. J Comp Physiol A 192: 613–624.P. K. StoddardH. H. ZakonM. R. MarkhamL. McAnelly2006Regulation and modulation of electric waveforms in gymnotiform electric fish.J Comp Physiol A192613624
- 30. Markham M. R, McAnelly M. L, Stoddard P. K, Zakon H. H (2009) Circadian and social cues regulate ion channel trafficking. PloS Biol 7(9): e1000203.M. R. MarkhamM. L. McAnellyP. K. StoddardH. H. Zakon2009Circadian and social cues regulate ion channel trafficking.PloS Biol7(9)e1000203
- 31. Singley J. A, Chavin W (1976) The diel rhythm of circulating ACTH titer in the goldfish (Carassius auratus l.). Comp Biochem Physiol A 53: 291–293.J. A. SingleyW. Chavin1976The diel rhythm of circulating ACTH titer in the goldfish (Carassius auratus l.).Comp Biochem Physiol A53291293
- 32. Markham M. R, Stoddard P. K (2005) Adrenocorticotropic hormone enhances the masculinity of an electric communication signal by modulating the waveform and timing of action potentials within individual cells. J Neurosci 25: 8746–8754.M. R. MarkhamP. K. Stoddard2005Adrenocorticotropic hormone enhances the masculinity of an electric communication signal by modulating the waveform and timing of action potentials within individual cells.J Neurosci2587468754
- 33. Sheng M, Lee S. H (2001) AMPA receptor trafficking and the control of synaptic transmission. Cell 105: 825–828.M. ShengS. H. Lee2001AMPA receptor trafficking and the control of synaptic transmission.Cell105825828
- 34. Burrone J, Murthy V. N (2001) Synaptic plasticity: rush hour traffic in the AMPA lanes. Curr Biol 11: R274–277.J. BurroneV. N. Murthy2001Synaptic plasticity: rush hour traffic in the AMPA lanes.Curr Biol11R274277
- 35. Wan Q, Xiong Z. G, Man H. Y, Ackerley C. A, Braunton J, et al. (1997) Recruitment of functional GABA(A) receptors to postsynaptic domains by insulin. Nature 388: 686–690.Q. WanZ. G. XiongH. Y. ManC. A. AckerleyJ. Braunton1997Recruitment of functional GABA(A) receptors to postsynaptic domains by insulin.Nature388686690
- 36. Strand C. R, Ross M. S, Weiss S. L, Deviche P (2008) Testosterone and social context affect singing behavior but not song control region volumes in adult male songbirds in the fall. Behav Processes 78: 29–37.C. R. StrandM. S. RossS. L. WeissP. Deviche2008Testosterone and social context affect singing behavior but not song control region volumes in adult male songbirds in the fall.Behav Processes782937