Harmonin enhances voltage-dependent facilitation of Cav1.3 channels and synchronous exocytosis in mouse inner hair cells

Gregory FD, Pangrsic T, Calin-Jageman IE, Moser T, Lee A

J. Physiol. (Lond.) 2013 Jul;591(Pt 13):3253-69

PMID: 23613530


Cav1.3 channels mediate Ca(2+) influx that triggers exocytosis of glutamate at cochlear inner hair cell (IHC) synapses. Harmonin is a PDZ-domain-containing protein that interacts with the C-terminus of the Cav1.3 α1 subunit (α11.3) and controls cell surface Cav1.3 levels by promoting ubiquitin-dependent proteosomal degradation. However, PDZ-domain-containing proteins have diverse functions and regulate other Cav1.3 properties, which could collectively influence presynaptic transmitter release. Here, we report that harmonin binding to the α11.3 distal C-terminus (dCT) enhances voltage-dependent facilitation (VDF) of Cav1.3 currents both in transfected HEK293T cells and in mouse inner hair cells. In HEK293T cells, this effect of harmonin was greater for Cav1.3 channels containing the auxiliary Cav β1 than with the β2 auxiliary subunit. Cav1.3 channels lacking the α11.3 dCT were insensitive to harmonin modulation. Moreover, the ‘deaf-circler’ dfcr mutant form of harmonin, which does not interact with the α11.3 dCT, did not promote VDF. In mature IHCs from mice expressing the dfcr harmonin mutant, Cav1.3 VDF was less than in control IHCs. This difference was not observed between control and dfcr IHCs prior to hearing onset. Membrane capacitance recordings from dfcr IHCs revealed a role for harmonin in synchronous exocytosis and in increasing the efficiency of Ca(2+) influx for triggering exocytosis. Collectively, our results indicate a multifaceted presynaptic role of harmonin in IHCs in regulating Cav1.3 Ca(2+) channels and exocytosis.

Glutamate transporters EAAT4 and EAAT5 are expressed in vestibular hair cells and calyx endings

Dalet A, Bonsacquet J, Gaboyard-Niay S, Calin-Jageman I, Chidavaenzi RL, Venteo S, Desmadryl G, Goldberg JM, Lysakowski A, Chabbert C

PLoS ONE 2012;7(9):e46261

PMID: 23049999


Glutamate is the neurotransmitter released from hair cells. Its clearance from the synaptic cleft can shape neurotransmission and prevent excitotoxicity. This may be particularly important in the inner ear and in other sensory organs where there is a continually high rate of neurotransmitter release. In the case of most cochlear and type II vestibular hair cells, clearance involves the diffusion of glutamate to supporting cells, where it is taken up by EAAT1 (GLAST), a glutamate transporter. A similar mechanism cannot work in vestibular type I hair cells as the presence of calyx endings separates supporting cells from hair-cell synapses. Because of this arrangement, it has been conjectured that a glutamate transporter must be present in the type I hair cell, the calyx ending, or both. Using whole-cell patch-clamp recordings, we demonstrate that a glutamate-activated anion current, attributable to a high-affinity glutamate transporter and blocked by DL-TBOA, is expressed in type I, but not in type II hair cells. Molecular investigations reveal that EAAT4 and EAAT5, two glutamate transporters that could underlie the anion current, are expressed in both type I and type II hair cells and in calyx endings. EAAT4 has been thought to be expressed almost exclusively in the cerebellum and EAAT5 in the retina. Our results show that these two transporters have a wider distribution in mice. This is the first demonstration of the presence of transporters in hair cells and provides one of the few examples of EAATs in presynaptic elements.

Harmonin inhibits presynaptic Cav1.3 Ca²⁺ channels in mouse inner hair cells

Gregory FD, Bryan KE, Pangršič T, Calin-Jageman IE, Moser T, Lee A

Nat. Neurosci. 2011 Sep;14(9):1109-11

PMID: 21822269


Harmonin is a scaffolding protein that is required for normal mechanosensory function in hair cells. We found a presynaptic association of harmonin and Ca(v)1.3 Ca(2+) channels at the mouse inner hair cell synapse, which limits channel availability through a ubiquitin-dependent pathway.

Molecular microdomains in a sensory terminal, the vestibular calyx ending

Lysakowski A, Gaboyard-Niay S, Calin-Jageman I, Chatlani S, Price SD, Eatock RA

J. Neurosci. 2011 Jul;31(27):10101-14

PMID: 21734302


Many primary vestibular afferents form large cup-shaped postsynaptic terminals (calyces) that envelope the basolateral surfaces of type I hair cells. The calyceal terminals both respond to glutamate released from ribbon synapses in the type I cells and initiate spikes that propagate to the afferent’s central terminals in the brainstem. The combination of synaptic and spike initiation functions in these unique sensory endings distinguishes them from the axonal nodes of central neurons and peripheral nerves, such as the sciatic nerve, which have provided most of our information about nodal specializations. We show that rat vestibular calyces express an unusual mix of voltage-gated Na and K channels and scaffolding, cell adhesion, and extracellular matrix proteins, which may hold the ion channels in place. Protein expression patterns form several microdomains within the calyx membrane: a synaptic domain facing the hair cell, the heminode abutting the first myelinated internode, and one or two intermediate domains. Differences in the expression and localization of proteins between afferent types and zones may contribute to known variations in afferent physiology.

Ca(v)1 L-type Ca2+ channel signaling complexes in neurons

Calin-Jageman I, Lee A

J. Neurochem. 2008 May;105(3):573-83

PMID: 18266933


Ca(v)1 L-type Ca2+ channels play crucial and diverse roles in the nervous system. The pre- and post-synaptic functions of Ca(v)1 channels not only depend on their intrinsic biophysical properties but also their dynamic regulation by a host of cellular influences. These include protein kinases and phosphatases, G-protein coupled receptors, scaffolding proteins, and Ca2+-binding proteins. The cytoplasmic domains of the main pore forming alpha(1) subunit of Ca(v)1 offer a number of binding sites for these modulators, permitting fast and localized regulation of Ca2+ entry. Through effects on Ca(v)1 gating, localization, and coupling to effectors, protein modulators are efficiently positioned to adjust Ca(v)1 Ca2+ signals that control neuronal excitability, synaptic plasticity, and gene expression.

Ca2+-binding proteins tune Ca2+-feedback to Cav1.3 channels in mouse auditory hair cells

Cui G, Meyer AC, Calin-Jageman I, Neef J, Haeseleer F, Moser T, Lee A

J. Physiol. (Lond.) 2007 Dec;585(Pt 3):791-803

PMID: 17947313


Sound coding at the auditory inner hair cell synapse requires graded changes in neurotransmitter release, triggered by sustained activation of presynaptic Ca(v)1.3 voltage-gated Ca(2+) channels. Central to their role in this regard, Ca(v)1.3 channels in inner hair cells show little Ca(2+)-dependent inactivation, a fast negative feedback regulation by incoming Ca(2+) ions, which depends on calmodulin association with the Ca(2+) channel alpha(1) subunit. Ca(2+)-dependent inactivation characterizes nearly all voltage-gated Ca(2+) channels including Ca(v)1.3 in other excitable cells. The mechanism underlying the limited autoregulation of Ca(v)1.3 in inner hair cells remains a mystery. Previously, we established calmodulin-like Ca(2+)-binding proteins in the brain and retina (CaBPs) as essential modulators of voltage-gated Ca(2+) channels. Here, we demonstrate that CaBPs differentially modify Ca(2+) feedback to Ca(v)1.3 channels in transfected cells and explore their significance for Ca(v)1.3 regulation in inner hair cells. Of multiple CaBPs detected in inner hair cells (CaBP1, CaBP2, CaBP4 and CaBP5), CaBP1 most efficiently blunts Ca(2+)-dependent inactivation of Ca(v)1.3. CaBP1 and CaBP4 both interact with calmodulin-binding sequences in Ca(v)1.3, but CaBP4 more weakly inhibits Ca(2+)-dependent inactivation than CaBP1. Ca(2+)-dependent inactivation is marginally greater in inner hair cells from CaBP4(-/-) than from wild-type mice, yet CaBP4(-/-) mice are not hearing-impaired. In contrast to CaBP4, CaBP1 is strongly localized at the presynaptic ribbon synapse of adult inner hair cells both in wild-type and CaBP4(-/-) mice and therefore is positioned to modulate native Ca(v)1.3 channels. Our results reveal unexpected diversity in the strengths of CaBPs as Ca(2+) channel modulators, and implicate CaBP1 rather than CaBP4 in conferring the anomalous slow inactivation of Ca(v)1.3 Ca(2+) currents required for auditory transmission.

Erbin enhances voltage-dependent facilitation of Ca(v)1.3 Ca2+ channels through relief of an autoinhibitory domain in the Ca(v)1.3 alpha1 subunit

Calin-Jageman I, Yu K, Hall RA, Mei L, Lee A

J. Neurosci. 2007 Feb;27(6):1374-85

PMID: 17287512


Ca(v)1.3 (L-type) voltage-gated Ca2+ channels have emerged as key players controlling Ca2+ signals at excitatory synapses. Compared with the more widely expressed Ca(v)1.2 L-type channel, relatively little is known about the mechanisms that regulate Ca(v)1.3 channels. Here, we describe a new role for the PSD-95 (postsynaptic density-95)/Discs large/ZO-1 (zona occludens-1) (PDZ) domain-containing protein, erbin, in directly potentiating Ca(v)1.3. Erbin specifically forms a complex with Ca(v)1.3, but not Ca(v)1.2, in transfected cells. The significance of erbin/Ca(v)1.3 interactions is supported by colocalization in somatodendritic domains of cortical neurons in culture and coimmunoprecipitation from rat brain lysates. In electrophysiological recordings, erbin augments facilitation of Ca(v)1.3 currents by a conditioning prepulse, a process known as voltage-dependent facilitation (VDF). This effect requires a direct interaction of the erbin PDZ domain with a PDZ recognition site in the C-terminal domain (CT) of the long variant of the Ca(v)1.3 alpha1 subunit (alpha1 1.3). Compared with Ca(v)1.3, the Ca(v)1.3b splice variant, which lacks a large fraction of the alpha1 1.3 CT, shows robust VDF that is not further affected by erbin. When coexpressed as an independent entity with Ca(v)1.3b or Ca(v)1.3 plus erbin, the alpha1 1.3 CT strongly suppresses VDF, signifying an autoinhibitory function of this part of the channel. These modulatory effects of erbin, but not alpha1 1.3 CT, depend on the identity of the auxiliary Ca2+ channel beta subunit. Our findings reveal a novel mechanism by which PDZ interactions and alternative splicing of alpha1 1.3 may influence activity-dependent regulation of Ca(v)1.3 channels at the synapse.

Regulation of the preprotachykinin-I gene promoter through a protein kinase A-dependent, cyclic AMP response element-binding protein-independent mechanism

Calin-Jageman IE, Wang J, Bannon MJ

J. Neurochem. 2006 Apr;97(1):255-64

PMID: 16515544


Preprotachykinin-I (PPT) gene expression is regulated by a number of stimuli that signal through cyclic AMP (cAMP)-mediated pathways. In the present study, forskolin, an adenylyl cyclase stimulator, significantly increased PPT mRNA levels in PPT-expressing RINm5F cells, an effect paralleled by an increase in PPT promoter-luciferase reporter construct activity. The forskolin-induced stimulation of PPT transcription was protein kinase A dependent (PKA), as shown by blockade with the PKA inhibitor N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide. We found that the activation protein 1/cAMP response element (AP1/CRE) site centered at -196 relative to the transcription start site was important for basal and forskolin-induced PPT promoter activity. Because of the involvement of PKA and the similarity of the AP1/CRE element to consensus CRE sequences, we investigated the role of CRE-binding protein (CREB) in the regulation of the PPT promoter. Surprisingly, overexpression of a dominant-negative CREB (i.e. CREB-A) did not affect basal or forskolin-induced PPT promoter activity. Furthermore, binding of CREB to the PPT promoter AP1/CRE site was not demonstrable in electrophoretic mobility shift assays. Rather, our experiments suggested that c-Jun is a member of the complex that binds to this site. We conclude that, at least in RINm5F cells, cAMP-mediated up-regulation of PPT gene expression does not involve CREB or CREB-related transcription factor recruitment to the AP1/CRE site.

Brain-specific RGS9-2 is localized to the nucleus via its unique proline-rich domain

Bouhamdan M, Michelhaugh SK, Calin-Jageman I, Ahern-Djamali S, Bannon MJ

Biochim. Biophys. Acta 2004 May;1691(2-3):141-50

PMID: 15110994


Brain-specific regulator of G protein signaling 9 (RGS9-2) is a member of a family of proteins that can function as GTPase-activating proteins for heterotrimeric G proteins. In the present study, we examined the intracellular distribution of RGS9-2 in native brain tissue and transfected cells. Immunocytochemical and immunoblot experiments revealed an unexpectedly high proportion of RGS9-2 within the nuclei of forebrain neurons. A similar intracellular distribution was seen in transfected COS-7 cells. The RGS9 binding partner G(beta5) further enhanced the nuclear localization of RGS9-2, but did not affect the strongly cytoplasmic localization of RGS9-1, the retinal form of RGS9. Deletion construct analysis revealed that the unique polyproline-rich C-terminus of brain-specific RGS9-2 contains sequences necessary and sufficient to target RGS9 to the nucleus of COS-7 cells, as well as cultured striatal neurons. Furthermore, RGS9-2 transfection increased the transcriptional activity of a neuronal gene construct normally expressed in RGS9-positive neurons, suggesting that nuclear RGS9 directly or indirectly regulates transcription in vivo. The nuclear localization of RGS9-2 suggests a heretofore-unanticipated role for this brain-specific protein in transducing signals to the nuclei of forebrain neurons.

Characterization of a chlorella virus PBCV-1 encoded ribonuclease III

Zhang Y, Calin-Jageman I, Gurnon JR, Choi TJ, Adams B, Nicholson AW, Van Etten JL

Virology 2003 Dec;317(1):73-83

PMID: 14675626


Sequence analysis of the 330-kb genome of chlorella virus PBCV-1 revealed an open reading frame, A464R, which encodes a protein with 30-35% amino acid identity to ribonuclease III (RNase III) from many bacteria. The a464r gene was cloned and the protein was expressed in Escherichia coli using the chitin-binding intein system. The recombinant PBCV-1 RNase III cleaves model dsRNA substrates, in a Mg(2+)-dependent manner, into a defined set of products. The substrate cleavage specificity overlaps, but is nonidentical to that of E. coli RNase III. The a464r gene is expressed very early during PBCV-1 infection, within 5-10 min p.i. The RNase III protein appears at 15 min p.i. and disappears by 120 min p.i. The a464r gene is highly conserved among the chlorella viruses. Phylogenetic analyses indicate that the PBCV enzyme is most closely related to Mycoplasma pneumoniae RNase III.