Irina E. Calin-Jageman

Cav1 L-type Ca2+ channel signaling complexes in neurons

Cav1 L‐type Ca2+ channels play crucial and diverse roles in the nervous system. The pre‐ and post‐synaptic functions of Cav1 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 α1 subunit of Cav1 offer a number of binding sites for these modulators, permitting fast and localized regulation of Ca2+ entry. Through effects on Cav1 gating, localization, and coupling to effectors, protein modulators are efficiently positioned to adjust Cav1 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

Sound coding at the auditory inner hair cell synapse requires graded changes in neurotransmitter release, triggered by sustained activation of presynaptic Cav1.3 voltage‐gated Ca2+ channels. Central to their role in this regard, Cav1.3 channels in inner hair cells show little Ca2+‐dependent inactivation, a fast negative feedback regulation by incoming Ca2+ ions, which depends on calmodulin association with the Ca2+ channel α1 subunit. Ca2+‐dependent inactivation characterizes nearly all voltage‐gated Ca2+ channels including Cav1.3 in other excitable cells. The mechanism underlying the limited autoregulation of Cav1.3 in inner hair cells remains a mystery. Previously, we established calmodulin‐like Ca2+‐binding proteins in the brain and retina (CaBPs) as essential modulators of voltage‐gated Ca2+ channels. Here, we demonstrate that CaBPs differentially modify Ca2+ feedback to Cav1.3 channels in transfected cells and explore their significance for Cav1.3 regulation in inner hair cells. Of multiple CaBPs detected in inner hair cells (CaBP1, CaBP2, CaBP4 and CaBP5), CaBP1 most efficiently blunts Ca2+‐dependent inactivation of Cav1.3. CaBP1 and CaBP4 both interact with calmodulin‐binding sequences in Cav1.3, but CaBP4 more weakly inhibits Ca2+‐dependent inactivation than CaBP1. Ca2+‐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 Cav1.3 channels. Our results reveal unexpected diversity in the strengths of CaBPs as Ca2+ channel modulators, and implicate CaBP1 rather than CaBP4 in conferring the anomalous slow inactivation of Cav1.3 Ca2+ currents required for auditory transmission.

Molecular Microdomains in a Sensory Terminal, the Vestibular Calyx Ending

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 afferents 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.

Escherichia coli Ribonuclease III: Affinity Purification of Hexahistidine-Tagged Enzyme and Assays for Substrate Binding and Cleavage

It is now evident that members of the RNase III family of nucleases have central roles in prokaryotic and eukaryotic RNA maturation and decay pathways. Ongoing research is uncovering new roles for RNase III homologs. For example, the phenomena of RNA interference (RNAi) and posttranscriptional gene silencing (PTGS) involve dsRNA processing, carried out by an RNase III homolog. We anticipate an increased focus on the mechanism, regulation, and biological roles of RNase III orthologs. Although the differences in the physicochemical properties of RNase III orthologs, and distinct substrate reactivity epitopes and ionic requirements for optimal activity, may mean that the protocols describe here are not strictly transferrable, the affinity purification methodology, and substrate preparation and use should be generally applicable.

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

Harmonin is a scaffolding protein that is required for normal mechanosensory function in hair cells. We found a presynaptic association of harmonin and Cav1.3 Ca2+ channels at the mouse inner hair cell synapse, which limits channel availability through a ubiquitin-dependent pathway.

Erbin Enhances Voltage-Dependent Facilitation of Cav1.3 Ca2+ Channels through Relief of an Autoinhibitory Domain in the Cav1.3 α1 Subunit

Cav1.3 (L-type) voltage-gated Ca2+ channels have emerged as key players controlling Ca2+ signals at excitatory synapses. Compared with the more widely expressed Cav1.2 L-type channel, relatively little is known about the mechanisms that regulate Cav1.3 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 Cav1.3. Erbin specifically forms a complex with Cav1.3, but not Cav1.2, in transfected cells. The significance of erbin/Cav1.3 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 Cav1.3 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 Cav1.3 α1 subunit (α11.3). Compared with Cav1.3, the Cav1.3b splice variant, which lacks a large fraction of the α11.3 CT, shows robust VDF that is not further affected by erbin. When coexpressed as an independent entity with Cav1.3b or Cav1.3 plus erbin, the α11.3 CT strongly suppresses VDF, signifying an autoinhibitory function of this part of the channel. These modulatory effects of erbin, but not α11.3 CT, depend on the identity of the auxiliary Ca2+ channel β subunit. Our findings reveal a novel mechanism by which PDZ interactions and alternative splicing of α11.3 may influence activity-dependent regulation of Cav1.3 channels at the synapse.

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

•  Cav1.3 Ca2+ channels mediate sound transmission by triggering presynaptic exocytosis of glutamate from cochlear inner hair cells (IHCs). •  Harmonin is a PDZ‐domain‐containing protein in IHCs that is altered in Usher syndrome, a form of deaf–blindness in humans. •  We show that harmonin enhances Cav1.3 voltage‐dependent facilitation (VDF) in transfected HEK293T cells in a manner that depends on the identity of the auxiliary Ca2+ channel β subunit. •  Cav1.3 VDF is impaired, and synchronous exocytosis and the Ca2+ efficiency of exocytosis are reduced, in IHCs from deaf‐circler mice expressing a mutant form of harmonin (dfcr) that cannot interact with Cav1.3. •  We conclude that harmonin regulates presynaptic function in mouse IHCs, which adds to our understanding of the factors that may influence hearing impairment in Usher syndrome.

Glutamate Transporters EAAT4 and EAAT5 Are Expressed in Vestibular Hair Cells and Calyx Endings

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.

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

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.

An Aplysia Egr homolog is rapidly and persistently regulated by long-term sensitization training

The Egr family of transcription factors plays a key role in long-term plasticity and memory in a number of vertebrate species. Here we identify and characterize ApEgr (GenBank: KC608221), an Egr homolog in the marine mollusk Aplysia californica. ApEgr codes for a predicted 593-amino acid protein with the highly conserved trio of zinc-fingered domains in the C-terminus that characterizes the Egr family of transcription factors. Promoter analysis shows that the ApEgr protein selectively recognizes the GSG motif recognized by vertebrate Egrs. Like mammalian Egrs, ApEgr is constitutively expressed in a range of tissues, including the CNS. Moreover, expression of ApEgr is bi-directionally regulated by changes in neural activity. Of most interest, the association between ApEgr function and memory may be conserved in Aplysia, as we observe rapid and long-lasting up-regulation of expression after long-term sensitization training. Taken together, our results suggest that Egrs may have memory functions that are conserved from mammals to mollusks.