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Pickpocket1 Is an Ionotropic Molecular Sensory Transducer*

  • Nina Boiko
    Affiliations
    Department of Physiology, University of Texas Health Sciences Center, San Antonio, Texas 78229
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  • Volodymyr Kucher
    Affiliations
    Department of Physiology, University of Texas Health Sciences Center, San Antonio, Texas 78229
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  • James D. Stockand
    Footnotes
    Affiliations
    Department of Physiology, University of Texas Health Sciences Center, San Antonio, Texas 78229
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  • Benjamin A. Eaton
    Correspondence
    To whom correspondence should be addressed: Univ. of Texas Health Sciences Center, Dept. of Physiology, 7703 Floyd Curl Dr., San Antonio, TX 78229. Tel.: 210-567-4383; Fax: 210-567-4410
    Footnotes
    Affiliations
    Department of Physiology, University of Texas Health Sciences Center, San Antonio, Texas 78229
    Search for articles by this author
  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant RO1 NS062811 (to B. A. E.). This work was also supported by American Heart Association Established Investigator Award 0640054N (to J. D. S.).
    1 These authors contributed equally to this work.
Open AccessPublished:October 01, 2012DOI:https://doi.org/10.1074/jbc.M112.411736
      The molecular transformation of an external stimulus into changes in sensory neuron activity is incompletely described. Although a number of molecules have been identified that can respond to stimuli, evidence that these molecules can transduce stimulation into useful neural activity is lacking. Here we demonstrate that pickpocket1 (ppk1), a Drosophila homolog of mammalian Degenerin/epithelial sodium channels, encodes an acid-sensing sodium channel that conducts a transient depolarizing current in multidendritic sensory neurons of Drosophila melanogaster. Stimulation of Ppk1 is sufficient to bring these sensory neurons to threshold, eliciting a burst of action potentials. The transient nature of the neural activity produced by Ppk1 activation is the result of Ppk1 channel gating properties. This model is supported by the observation of enhanced bursting activity in neurons expressing a gain of function ppk1 mutant harboring the degenerin mutation. These findings demonstrate that Ppk1 can function as an ionotropic molecular sensory transducer capable of transforming the perception of a stimulus into phasic neuronal activity in sensory neurons.

      Introduction

      Peripheral sensory neurons are capable of sensing and responding to a broad range of stimuli including mechanical, chemical, and thermal cues. These neurons are thought to express molecular sensory transducers that transform stimuli into changes in neuronal activity. For a protein to function as a molecular sensory transducer, it must not only be able to sense and respond to a stimulus but also be able to transform the sensing of the stimulus into an electrical signal capable of evoking the firing of action potentials in the sensory neuron.
      Ion channel proteins are particularly strong candidates to serve as molecular sensory transducers because their ion channel function enables them to transform the sensing of a stimulus, upon activation, into an electrical current that can alter neuron activity. Although there are many examples of ion channel receptors that can respond to sensory stimuli (
      • Coste B.
      • Mathur J.
      • Schmidt M.
      • Earley T.J.
      • Ranade S.
      • Petrus M.J.
      • Dubin A.E.
      • Patapoutian A.
      Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels.
      ,
      • Kim S.E.
      • Coste B.
      • Chadha A.
      • Cook B.
      • Patapoutian A.
      The role of Drosophila Piezo in mechanical nociception.
      ,
      • Geffeney S.L.
      • Cueva J.G.
      • Glauser D.A.
      • Doll J.C.
      • Lee T.H.
      • Montoya M.
      • Karania S.
      • Garakani A.M.
      • Pruitt B.L.
      • Goodman M.B.
      DEG/ENaC but not TRP channels are the major mechanoelectrical transduction channels in a C. elegans nociceptor.
      ,
      • O'Hagan R.
      • Chalfie M.
      • Goodman M.B.
      The MEC-4 DEG/ENaC channel of Caenorhabditis elegans touch receptor neurons transduces mechanical signals.
      ,
      • Hong K.
      • Mano I.
      • Driscoll M.
      In vivo structure-function analyses of Caenorhabditis elegans MEC-4, a candidate mechanosensory ion channel subunit.
      ,
      • Chatzigeorgiou M.
      • Yoo S.
      • Watson J.D.
      • Lee W.H.
      • Spencer W.C.
      • Kindt K.S.
      • Hwang S.W.
      • Miller 3rd, D.M.
      • Treinin M.
      • Driscoll M.
      • Schafer W.R.
      Specific roles for DEG/ENaC and TRP channels in touch and thermosensation in C. elegans nociceptors.
      ,
      • Walker R.G.
      • Willingham A.T.
      • Zuker C.S.
      A Drosophila mechanosensory transduction channel.
      ,
      • Kang L.
      • Gao J.
      • Schafer W.R.
      • Xie Z.
      • Xu X.Z.
      C. elegans TRP family protein TRP-4 is a pore-forming subunit of a native mechanotransduction channel.
      ), there are few examples where activation of these channels can drive neuronal activity.
      The subgroup of mammalian Deg/ENaC
      The abbreviations used are: Deg
      Degenerin
      ENaC
      epithelial sodium channel
      ASIC
      acid-sensing ion channel
      md
      multidendritic
      TTX
      tetrodotoxin.
      channels activated by extracellular acidic pH represents attractive candidates for functioning in mammalian sensory neurons as molecular sensory transducers. These acid-sensing ion channels (ASICs) are expressed in sensory neurons, including in the dorsal root ganglion, and have been implicated in mechanosensation and proprioception (
      • Lingueglia E.
      Acid-sensing ion channels in sensory perception.
      ,
      • Price M.P.
      • McIlwrath S.L.
      • Xie J.
      • Cheng C.
      • Qiao J.
      • Tarr D.E.
      • Sluka K.A.
      • Brennan T.J.
      • Lewin G.R.
      • Welsh M.J.
      The DRASIC cation channel contributes to the detection of cutaneous touch and acid stimuli in mice.
      ,
      • Bianchi L.
      • Driscoll M.
      Protons at the gate. DEG/ENaC ion channels help us feel and remember.
      ,
      • Borzan J.
      • Zhao C.
      • Meyer R.A.
      • Raja S.N.
      A role for acid-sensing ion channel 3, but not acid-sensing ion channel 2, in sensing dynamic mechanical stimuli.
      ,
      • Lu Y.
      • Ma X.
      • Sabharwal R.
      • Snitsarev V.
      • Morgan D.
      • Rahmouni K.
      • Drummond H.A.
      • Whiteis C.A.
      • Costa V.
      • Price M.
      • Benson C.
      • Welsh M.J.
      • Chapleau M.W.
      • Abboud F.M.
      The ion channel ASIC2 is required for baroreceptor and autonomic control of the circulation.
      ,
      • Darboux I.
      • Lingueglia E.
      • Pauron D.
      • Barbry P.
      • Lazdunski M.
      A new member of the amiloride-sensitive sodium channel family in Drosophila melanogaster peripheral nervous system.
      ,
      • Benson C.J.
      • Xie J.
      • Wemmie J.A.
      • Price M.P.
      • Henss J.M.
      • Welsh M.J.
      • Snyder P.M.
      Heteromultimers of DEG/ENaC subunits form H+-gated channels in mouse sensory neurons.
      ). The Drosophila Deg/ENaC homolog, ppk1, is expressed in a restrictive manner in class IV multidentritic (md) sensory neurons (
      • Darboux I.
      • Lingueglia E.
      • Pauron D.
      • Barbry P.
      • Lazdunski M.
      A new member of the amiloride-sensitive sodium channel family in Drosophila melanogaster peripheral nervous system.
      ,
      • Ainsley J.A.
      • Pettus J.M.
      • Bosenko D.
      • Gerstein C.E.
      • Zinkevich N.
      • Anderson M.G.
      • Adams C.M.
      • Welsh M.J.
      • Johnson W.A.
      Enhanced locomotion caused by loss of the Drosophila DEG/ENaC protein Pickpocket1.
      ,
      • Adams C.M.
      • Anderson M.G.
      • Motto D.G.
      • Price M.P.
      • Johnson W.A.
      • Welsh M.J.
      Ripped pocket and pickpocket, novel Drosophila DEG/ENaC subunits expressed in early development and in mechanosensory neurons.
      ,
      • Zhong L.
      • Hwang R.Y.
      • Tracey W.D.
      Pickpocket is a DEG/ENaC protein required for mechanical nociception in Drosophila larvae.
      ). These polymodal sensory neurons form extensive dendritic networks that ramify beneath the epidermis and are required for normal proprioception and nociception (
      • Ainsley J.A.
      • Pettus J.M.
      • Bosenko D.
      • Gerstein C.E.
      • Zinkevich N.
      • Anderson M.G.
      • Adams C.M.
      • Welsh M.J.
      • Johnson W.A.
      Enhanced locomotion caused by loss of the Drosophila DEG/ENaC protein Pickpocket1.
      ,
      • Song W.
      • Onishi M.
      • Jan L.Y.
      • Jan Y.N.
      Peripheral multidendritic sensory neurons are necessary for rhythmic locomotion behavior in Drosophila larvae.
      ,
      • Hwang R.Y.
      • Zhong L.
      • Xu Y.
      • Johnson T.
      • Zhang F.
      • Deisseroth K.
      • Tracey W.D.
      Nociceptive neurons protect Drosophila larvae from parasitoid wasps.
      ). In consideration of such findings, we hypothesized that md neurons in Drosophila, in which Ppk1 is restrictively expressed, may harbor acid-sensitive channels that can function as ionotropic molecular sensory transducers.
      In the current study, we demonstrate that ppk1 encodes an acid-sensing ion channel in Drosophila class IV md sensory neurons. Activation of the transient depolarizing sodium current conducted by Ppk1 is sufficient to drive md neurons to threshold, eliciting a burst of stimulus-dependent action potentials. Furthermore, this burst of action potentials is sensitive to changes in gating because stimulation of a Ppk1 protein harboring the gain of function degenerin mutation results in sustained bursting and loss of normal sensory neuron function. Together, these findings demonstrate that Ppk1 can function as a physiologically relevant ionotropic molecular sensory transducer capable of transforming stimuli into changes in neuronal activity.

      DISCUSSION

      The current results demonstrate that the Drosophila Deg/ENaC protein, Ppk1, is a key component of an acid-sensing ion channel expressed in class IV md sensory neurons. Activated Ppk1 channels conduct a transient depolarizing sodium current in md neurons. Ppk1 is not active at rest, however, and consequently contributes little to the resting membrane potential, the shape of the action potential, and the inherent excitability of md neurons. Rather, stimulus-dependent activation of Ppk1 channels drives md neurons to threshold evoking a phasic burst of action potentials. In this regard, Ppk1 channels serve as ionotropic molecular sensory transducers functioning as incidence detectors capable of transforming an external stimulus into an electrical signal able to evoke action potential firing in class IV md neurons. This function of Ppk1 (in md neurons) is critical for normal sensory perception.

      Ppk1 Is a Critical Component of an Acid-sensing Deg/ENaC Channel

      The results from the current electrophysiology studies provide compelling evidence that Ppk1 is a key component of an ion channel in md neurons: normal ppk1 expression and function are required for a transient, acid-sensing, amiloride-sensitive depolarizing sodium current in these sensory neurons. In this regard, this Drosophila homolog mirrors the ion channel function of other members of the Deg/ENaC channel family, in particular, the mammalian ASIC and Caenorhabditis elegans degenerin channel proteins also expressed in sensory neurons (
      • Lingueglia E.
      Acid-sensing ion channels in sensory perception.
      ,
      • Bianchi L.
      • Driscoll M.
      Protons at the gate. DEG/ENaC ion channels help us feel and remember.
      ,
      • Mano I.
      • Driscoll M.
      DEG/ENaC channels. A touchy superfamily that watches its salt.
      ). Sequence homology and structural similarity with ASIC proteins, which contribute to a conductive pore and for which the structure has been resolved (
      • Jasti J.
      • Furukawa H.
      • Gonzales E.B.
      • Gouaux E.
      Structure of acid-sensing ion channel 1 at 1.9 A resolution and low pH.
      ,
      • Gonzales E.B.
      • Kawate T.
      • Gouaux E.
      Pore architecture and ion sites in acid-sensing ion channels and P2X receptors.
      ), are consistent with Ppk1 being a pore-forming subunit. This is supported by our findings that a gain of function mutation in Ppk1 changes the biophysical properties of the transient current associated with the expression and function of this channel protein.
      Importantly, the alteration in gating by the gain of function mutation was sufficient to alter sensory responses, consistent with Ppk1 functioning during signal transduction in sensory neurons. Moreover, the observation that the behavioral phenotype of larvae harboring a gain of function ppk1 mutation in their md neurons phenocopies larvae harboring loss of function ppk1 mutations is similar to observations made for loss and gain of function mutations in Deg/ENaC channels expressed in C. elegans touch receptors (
      • Hong K.
      • Driscoll M.
      A transmembrane domain of the putative channel subunit MEC-4 influences mechanotransduction and neurodegeneration in C. elegans.
      ,
      • Driscoll M.
      • Chalfie M.
      The mec-4 gene is a member of a family of Caenorhabditis elegans genes that can mutate to induce neuronal degeneration.
      ,
      • Chalfie M.
      • Wolinsky E.
      The identification and suppression of inherited neurodegeneration in Caenorhabditis elegans.
      ,
      • Huang M.
      • Chalfie M.
      Gene interactions affecting mechanosensory transduction in Caenorhabditis elegans.
      ). These results support that any change in normal Ppk1 activity, be it an increase or a decrease, disrupts the ability of md neurons to transform sensory information appropriately, compromising normal behavioral responses. This emphasizes the importance of the appropriate activation of Deg/ENaC channels to the process of changing a stimulus into a properly graded electrical signal during sensory transduction.

      Deg/ENaC Channels Function as Molecular Signal Transducers

      The current studies demonstrate that decreases in pH activate and then quickly inactivate Ppk1 channels. This is similar to the effects of decreases in pH on mammalian ASIC channels (
      • Lingueglia E.
      Acid-sensing ion channels in sensory perception.
      ,
      • Price M.P.
      • McIlwrath S.L.
      • Xie J.
      • Cheng C.
      • Qiao J.
      • Tarr D.E.
      • Sluka K.A.
      • Brennan T.J.
      • Lewin G.R.
      • Welsh M.J.
      The DRASIC cation channel contributes to the detection of cutaneous touch and acid stimuli in mice.
      ,
      • Benson C.J.
      • Xie J.
      • Wemmie J.A.
      • Price M.P.
      • Henss J.M.
      • Welsh M.J.
      • Snyder P.M.
      Heteromultimers of DEG/ENaC subunits form H+-gated channels in mouse sensory neurons.
      ) with the exception that the inactivation of Ppk1 is particularly rapid. Importantly, the ability to relieve activated Ppk1 from blockade following stimulation by an acid challenge allowed us to test whether the activation of Deg/ENaC proteins is sufficient to drive sensory neurons to threshold and thus function as ionotropic molecular sensory transducers. These results help define the role that Deg/ENaC channels play in the mechanistic and ionic basis of sensory transduction. They serve two discrete functions: 1) during the initial phase of sensory transduction, they act as protein sensors involved in the direct perception of the stimulus, and 2) through their ability to conduct a depolarizing current upon stimulus-dependent activation capable of bringing the membrane potential to threshold, they transform the sensing of a stimulus into an electrical signal that results in changes in neuronal activity. Interestingly, the neuronal activity resulting from the activation of Ppk1 is shaped by the gating properties of the channel, suggesting that these channels can also play an important role in the encoding of the stimuli. This idea is supported by the observation of the deleterious effects of the degenerin mutation on mechanosensory behavior.
      The current results provide support for the idea that Deg/ENaC channels function as ionotropic molecular sensory transducers important for sensory transduction in peripheral sensory neurons. This solidly places Deg/ENaC channels, along with TRP and Piezo channels (
      • Coste B.
      • Mathur J.
      • Schmidt M.
      • Earley T.J.
      • Ranade S.
      • Petrus M.J.
      • Dubin A.E.
      • Patapoutian A.
      Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels.
      ,
      • Kim S.E.
      • Coste B.
      • Chadha A.
      • Cook B.
      • Patapoutian A.
      The role of Drosophila Piezo in mechanical nociception.
      ,
      • Geffeney S.L.
      • Cueva J.G.
      • Glauser D.A.
      • Doll J.C.
      • Lee T.H.
      • Montoya M.
      • Karania S.
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      • Goodman M.B.
      DEG/ENaC but not TRP channels are the major mechanoelectrical transduction channels in a C. elegans nociceptor.
      ,
      • Walker R.G.
      • Willingham A.T.
      • Zuker C.S.
      A Drosophila mechanosensory transduction channel.
      ,
      • Kang L.
      • Gao J.
      • Schafer W.R.
      • Xie Z.
      • Xu X.Z.
      C. elegans TRP family protein TRP-4 is a pore-forming subunit of a native mechanotransduction channel.
      ,
      • Cheng L.E.
      • Song W.
      • Looger L.L.
      • Jan L.Y.
      • Jan Y.N.
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      ,
      • Li W.
      • Feng Z.
      • Sternberg P.W.
      • Xu X.Z.
      A C. elegans stretch receptor neuron revealed by a mechanosensitive TRP channel homologue.
      ), into a small group of ion channels capable of sensing and transforming stimuli into electrical signals. The current results extend understanding significantly by identifying Deg/ENaC channel proteins as essential elements in sensory transduction, being important in both the early formative events encoding sensory stimuli and the transformative events resulting in changes in sensory neuron activity upon channel activation. The mechanistic paradigm emerging from the current studies, in consideration of earlier findings regarding the function of Deg/ENaC channels, has stimulus-dependent activation of Deg/ENaC channels in sensory dendrites, resulting in a depolarization of the plasma membrane via a transient inward sodium current. The resulting transient depolarization to threshold evokes a burst of action potentials, defined by channel gating properties, resulting in the transformation of the stimulus into an electrical signal in sensory neurons that carries instructive information into the nervous system, allowing for the appropriate behavioral response.

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