Voltage-gated Ca_v1 channels in disorders of vision and hearing

Abstract

Ca_v1 channels mediate L-type Ca²⁺ currents that trigger the exocytotic release of glutamate from the specialized “ribbon” synapse of retinal photoreceptors (PRs) and cochlear inner hair cells (IHCs). Genetic evidence from animal models and humans support a role for Ca_v1.3 and Ca_v1.4 as the primary Ca_v channels in IHCs and PRs, respectively. Because of the unique features of transmission at ribbon synapses, Ca_v1.3 and Ca_v1.4 exhibit unusual properties that are well-suited for their physiological roles. These properties may be intrinsic to the channel subunit(s) and/or may be conferred by regulatory interactions with synaptic signaling molecules. This review will cover advances in our understanding of the function of Ca_v1 channels at sensory ribbon synapses, and how dysregulation of these channels leads to disorders of vision and hearing.

Graphical abstract

Cav1.3 and Cav1.4 channel complexes of inner hair cells (IHC) and photoreceptor (PR) cells, respectively, share a number of biophysical properties and modulatory proteins at ribbon synapses (r = ribbon, sv = synaptic vesicles).

Introduction

The perception of light and sound is initiated by photoreceptors (PRs) in the retina and inner hair cells (IHCs) in the cochlea, respectively. Both PRs and IHCs convert an environmental stimulus into an electrical signal that is communicated to second-order neurons and ultimately to the brain. An important structural feature of both PRs and IHCs is the synaptic ribbon. Associated with the presynaptic active zone, the ribbon tethers thousands of synaptic vesicles and allows for rapid and sustained release of glutamate in response to graded changes in membrane potential [1]. Voltage-gated Ca²⁺ channels are positioned within nanometers of the ribbon [2], allowing for tight coupling of depolarization-dependent Ca²⁺ influx and the molecular machinery involved in exocytosis. In contrast to the dominant role of Ca_v2 channels at most synapses in the central nervous system, Ca_v1 channels are required for exocytosis at ribbon synapses [3-5]. Visual and acoustic stimuli must be encoded continuously with high fidelity and sensitivity. This review will highlight the unique features and regulatory functions of Ca_v1 channels at the PR and IHC synapse, and their importance for faithful transmission of sensory stimuli.

Ca_v1.3 channels and hearing

In the cochlea, sound-induced vibrations displace hair bundles rooted in the apical surface of IHCs. The subsequent modulation of mechanoelectrical transduction currents causes a graded receptor potential that regulates the opening of Ca_v1 channels clustered near the presynaptic ribbon. Ca²⁺ influx through these channels triggers the exocytotic release of glutamate onto postsynaptic spiral ganglion neuron afferents, which transmit auditory information into the central nervous system. Even in the absence of sensory stimulation, IHC synapses are tonically active, and support spontaneous afferent firing rates that can exceed 100 Hz [6]. The intensity and timing of sound is encoded by an increase in steady-state afferent firing rates, and by the ability of IHCs to phase-lock transmitter release to frequencies up to 5 kHz, respectively.

Current evidence favors a crucial role for Ca_v1.3 channels as the major Ca_v channel in IHCs. For example, in mice lacking Ca_v1.3 channels (Ca_v1.3 KO), IHCs are incapable of evoked exocytosis and exhibit whole-cell Ca²⁺ currents that are ∼90% of that in IHCs from wild-type mice [7, 8]. As a consequence, Ca_v1.3 KO mice are deaf, with no evidence of sound-evoked afferent activity in auditory brainstem responses (ABR) [7, 9]. Other components of the Ca_v1.3 complex in IHCs include the auxiliary Ca_v β [10] and α₂δ subunits.

Analyses of Ca_v1.3 channels in heterologous expression systems indicate a number of properties consistent with those of native Ca_v channels and their physiological demands in IHCs. First, Ca_v1.3 activates rapidly compared to the related Ca_v1.2 channel abundant in the brain and heart [11, 12]. Whole-cell and single-channel recordings of Ca²⁺ currents in IHCs indicate time constants for activation and latency to first opening in the submillisecond range [13-17]. Rapid activation kinetics of Ca_v1.3 channels are likely important for temporal aspects of sound coding, such as the rapid onset of sound and the ability to accurately trigger firing of the auditory nerve to reflect sound frequency (i.e., phase locking).

Second, Ca_v1.3 channels activate at relatively negative voltages [11, 12]. In single-channel recordings of immature mouse IHCs, channel openings were observed at voltages as negative as -70 mV [14]. Since the resting potential of these cells is ∼ -60 mV [18], the negative activation threshold of Cav1.3 would be able to support tonic transmitter release at rest. Moreover, the stronger activation of these channels in response to sound-evoked alterations in the receptor potential could encode increased sound intensity with greater rates of transmitter release.

Third, Ca_v1.3 channels in IHCs exhibit little Ca²⁺- or voltage- dependent inactivation (CDI, VDI), in contrast to their behavior in heterologous expression systems and in the heart [11, 16, 17, 19-23]. CDI is a negative feedback regulation mediated by permeating Ca²⁺ ions and relies on calmodulin (CaM) interacting with an IQ domain in the cytoplasmic C-terminal domain of the Ca_v1.3 α₁ subunit. CDI is not seen with Ba²⁺ as the charge carrier since Ba²⁺ binds poorly to CaM, so Ba²⁺ currents exhibit VDI, which is generally slower than CDI, (reviewed in [24]). The low levels of CDI in IHCs are thought to support the continuous encoding of sound information through sustained transmitter release. Possible factors contributing to the limited CDI of Ca_v1.3 channels in IHCs include the expression of Ca_v1.3 variants lacking a functional IQ domain [25]. In addition, the synaptic scaffolding protein, Rab3-interacting molecule 2 (RIM2), inhibits CDI and VDI of Ca_v1.3 channels in transfected tsA-201 cells. However, RIM2 is primarily expressed in the immature cochlea [26], such that other factors must contribute to the slow CDI and VDI in mature IHCs.

One possibility concerns CaBPs, a family of CaM-like Ca²⁺ binding proteins that are highly expressed in the brain, retina, and inner ear [20, 27-31]. Like CaM, CaBPs have 4 EF-hand Ca²⁺ binding domains, at least one of which is non-functional [27]. Antibodies against CaBP1, CaBP2, CaBP4, and CaBP5 label both immature and mature IHCs [20, 28]. In transfected HEK293T cells, CaBPs suppress CDI of Ca_v1.3 [20, 28, 32]. The mechanism may involve displacement of CaM by CaBPs from the Ca_v1 α₁ IQ domain [33-35]. However, CaBPs bind to additional sites in Ca_v1 α₁, and so may allosterically modulate CaM interactions with the channel [36-38]. CaBP2 may be an important physiological regulator of Ca_v1.3 in IHCs since a mutation in the CaBP2 gene causes moderate-to severe sensorineural hearing loss. The mutation causes premature truncation of CaBP2, such that the mutant CaBP2 lacks the C-terminal EF-hands. In transfected HEK293T cells, the mutant CaBP2 is less able to suppress CDI of Ca_v1.3 channels [39]. While additional research is required, increased CDI of Ca_v1.3 currents may be insufficient to support sustained neurotransmitter release from IHCs required for proper sound coding.

The requirement for Ca_v1.3 for hearing is clearly illustrated by the profound deafness seen in individuals with a mutation in CACNA1D [40]. This mutation results in a glycine insertion into the transmembrane helix IS6 in Ca_v1.3 variants containing exon 8B. In transfected tsA-201 cells, mutant channels are trafficked to the plasma membrane but do not conduct Ca²⁺ currents. The mechanism may involve reduced ability of voltage-sensors to couple to pore opening, or some failure of conductance downstream of pore-opening. In addition to deafness, the affected individuals exhibit severe sinus bradycardia [40], consistent with the role of Ca_v1.3 in regulating sinoatrial pacemaking and with the cardiac phenotype of Ca_v1.3 KO mice [7, 19].

Dysregulation of Ca_v1.3 channels is implicated in additional forms of hearing impairment. For example, severe hearing loss can result from thyroid hormone (TH) deficiency, mutations in thyroid hormone receptor β, or iodine deficiency [41]. Studies of TH-deficient rodents indicate abnormal maturation of IHC ionic currents and physiology. In normal mouse IHCs, Ca_v1.3 currents and BK K⁺ currents normally undergo a developmental decline or increase, respectively, prior to the onset of hearing in mice (postnatal day (P)12) [42, 43]. However, Ca_v1.3 currents remain elevated, and BK currents are undetectable, in TH-deficient rats and mice [44, 45]. The abnormally elevated Ca_v1.3 current density may result from alterations in Ca_v1.3 trafficking or turnover, since the corresponding mRNA levels are not affected by TH deficiency [44]. Due to the maintained high levels of Ca_v1.3, spontaneous Ca²⁺-dependent action potentials, which normally disappear around P13 [8, 46], are still evident in TH-deficient mice [44, 45]. IHCs from these mice also exhibit abnormalities in afferent and efferent innervation that suggest that IHCs have not transitioned at the molecular or morphological level to support tonic, graded changes in neurotransmitter release in response to sound stimulation [44]. TH is required for multiple aspects of auditory development, including maturation of the tectorial membrane and the generation of the endocochlear potential [41]. The extent to which the synaptic defects in IHC maturation contribute to deafness due to TH deficiency remains an open question.

A failure to regulate Ca_v1.3 channels also is found in a mouse model of Usher syndrome – the most common cause of combined deafness and blindness in humans [47]. “Deaf-circler” mice (dfcr) bear a mutation in the harmonin gene, which is a target of human mutations causing Usher syndrome [48]. Harmonin contains PDZ-domains, which are well-established motifs for protein-protein interactions [49]. Harmonin binds to a consensus PDZ binding site at the C-terminus of the Ca_v1.3 α₁ subunit, and this interaction inhibits Ca_v1.3 current density in transfected HEK293T cells. The mechanism involves enhanced proteosomal degradation of Ca_v1.3, which can be reversed by the proteosomal inhibitor MG132. The dfcr mutation prevents harmonin binding to Ca_v1.3 and causes increased Ca_v1.3 current density in dfcr compared to control IHCs [50]. Immunolabeling for harmonin at IHC synapses increases after hearing onset (P12) [50]. These results support a model in which harmonin promotes developmental trimming of Ca_v1.3 channels, which may be important for IHC maturation. Harmonin also enhances voltage-dependent facilitation of Ca_v1.3 channels [51], which may be important for boosting Ca_v1.3 function during sound stimulation. The primary cause of deafness associated with harmonin mutations is likely not to involve Ca_v1.3 channels given that harmonin is also a key regulatory of mechanosensory channels in IHC hair bundles [52]. However, synaptic abnormalities due to altered regulation of Ca_v1.3 trafficking and function could exacerbate the mechanosensory defect.

Ca_v1.4 channels and vision

In the retina, rod PRs mediate vision in dim light, while cone PRs mediate daytime vision and color perception. At the relatively depolarized membrane potential of PRs in darkness (∼ -40 mV), the opening of Ca_v1 channels clustered near the presynaptic ribbon triggers the sustained release of glutamate, which inhibits postsynaptic ON bipolar cells. Light-dependent hyperpolarization suppresses Ca_v1-dependent glutamate release, which depolarizes ON bipolar cells, ultimately enhancing the activity of third-order retinal ganglion cells that transmit visual information into the brain. Cone PRs form synapses with multiple types of ON or OFF bipolar neurons, which are either depolarized or hyperpolarized, respectively, by light stimulation of cones.

The major Ca_v1 channel at PR synapses is Ca_v1.4, which localizes in rod and cone PR terminals in the outer plexiform layer of the retina [53-57]. In mice with genetic inactivation of the Ca_v1.4 α₁ subunit (Ca_v1.4 KO), Ca²⁺ entry into PRs in response to depolarization is substantially impaired [58]. In electroretinograms, which measure visual function, Ca_v1.4 KO mice show no evidence of synaptic transmission between PRs and bipolar cells [56, 58]. However, Ca_v1.4 KO mice also exhibit PR degeneration with age [59] and defects in PR synapse morphology [58, 60] due to a failure in synapse maturation [56, 59, 61]. In addition to Ca_v1.4 α₁, the Ca_v1.4 complex at PR synapses likely includes auxiliary Ca_vβ₂ and α₂δ₄ subunits. Mice with reduced expression of Ca_vβ₂ and α₂δ₄ exhibit a similar visual and synaptic phenotype as in Ca_v1.4 KO mice [62, 63]. Immunochemical studies support the association of Ca_v1.4 α₁ with Ca_vβ₂ and α₂δ₄ in mouse retina [64].

Ca_v1.4 channels in heterologous expression systems exhibit properties similar to native Ca_v1 channels in PRs [65]. Compared to Ca_v1.2 channels, Ca_v1.4 channels activate at more negative voltages, which allows for tonic transmitter release at the dark membrane potential [66-68]. Although the IQ domain is conserved in Ca_v1.4 α₁, a C-terminal modulatory domain (CTM) of this channel inhibits CDI. The mechanism involves an intramolecular interaction between the CTM and a proximal region of the Ca_v1.4 α₁ C-terminal domain, which inhibits Ca²⁺-free (apo) CaM interactions with the IQ domain [69-72]. As for Ca_v1.3 channels at the IHC synapse, limited CDI of Ca_v1.4 channels at PR synapses supports the continuous release of glutamate that is required for sensory encoding.

In contrast to the presence of multiple CaBPs in IHCs, CaBP4 is the only CaBP family member expressed in PRs. CaBP4 interacts with and colocalizes with Ca_v1.4 in the outer plexiform layer of the mouse retina [29, 56, 64]. While CaBP4 does not regulate CDI of full-length Ca_v1.4 channels, deletion of the ICDI allows CaBP4 suppression of Ca_v1.4 CDI [73]. CaBP4 binds to the IQ domain in Ca_v1.4 α₁ and inhibits the intermolecular binding of the CTM with the proximal Ca_v1.4 α₁ C-terminal site [73]. In transfected HEK293T cells, CaBP4 enhances voltage-dependent activation and reduces VDI of Ca_v1.4 channels [29, 73]. The net increase in Ca_v1.4 channel availability due to CaBP4 may be important for facilitating glutamate release from PRs in darkness. Consistent with this hypothesis, mice lacking CaBP4 (CaBP4 KO) exhibit visual and retinal phenotypes similar to those in Ca_v1.4 KO mice. In addition to significantly impaired light-modulated PR transmission, the morphology of PR synapses is abnormal [29, 56].

A requirement for Ca_v1.4 in human vision is supported by the multiple visual disorders associated with mutations in the gene encoding Ca_v1.4 α₁, CACNA1F. The visual phenotypes associated with loss-of function of Ca_v1.4 are generally less severe in humans than in mouse models of the disease, which may be due to the normal or compensatory expression of Ca_v1.3 channels in human PRs. These disorders include congenital stationary night blindness type 2 (CSNB2), which causes heterogeneous symptoms that may include low visual acuity, myopia, and nystagmus [74-77]. Despite the name, night blindness is not always associated with CSNB2 phenotypes [78]. Other disorders associated with CACNA1F mutations include Åland island eye disease [79, 80] and cone-rod dystrophy (CORDX) [74, 81, 82]. Visual phenotypes of these disorders are similar to those in CSNB2, although there is early and more severe loss of cone function in CORDX compared to in CSNB2 [83]. An autosomal recessive form of cone-rod dystrophy is caused by mutations in the CACNA2D4 gene encoding α₂δ₄ [82], consistent with its importance as a Ca_v1.4 subunit [64].

The clinical variability associated with CACNA1F mutations may result from the functional impact of a particular mutation on Ca_v1.4 channels. In electrophysiological analyses in heterologous expression systems, CSNB2 mutant Ca_v1.4 channels may exhibit a loss-of function, gain-of function, and sometimes no observable difference compared to wild-type channels [68, 84]. For example, one CSNB2 mutation causes substitution of a proline for leucine860 (L860P), which is located in a conserved amphipathic helix in the loop connecting domains II and III. L860P reduces the number of function Ca_v1.4 channels at the cell surface by increasing the turnover of the protein [85]. While this would conceivably reduce Ca²⁺ signals supporting PR glutamate release in darkness, it is also possible that PR synapse development would be impaired, given that PR synapses do not form in Ca_v1.4 KO mice [56, 59, 61]. More complex mechanisms may result from truncating mutations that delete the CTM from Ca_v1.4 (K1591X [86], R1827X [87]). These mutations cause a loss-of function by increasing CDI, but also a gain-of function through enhance voltage-dependent activation, a process which the CTM normally suppresses[71, 85]. A mouse model of a gain-of function CSNB2 mutation (I745T) has provided some insights into the underlying pathophysiological mechanisms. This mutation causes an unusually severe form of CSNB2, and a large (-30 mV) shift in the voltage-dependence of Ca_v1.4 activation [88]. I745T knock-in mice exhibit abnormal PR synapse morphology, reduced cone and rod transmission, and degeneration of PRs due to apoptosis [55, 59].

Two mutations in the gene encoding CaBP4 also contribute to visual phenotypes similar to those in patients with CACNA1F mutations. One mutation is a valine substitution for glutamate and a frameshift that elongates the protein (E267fs). The second is a truncating mutation (R216X) that deletes the C-terminal EF-hands of CaBP4 [89, 90], which is essentially identical in nature to the CaBP2 mutation that causes autosomal recessive hearing loss [32]. These mutations do not affect the ability of CaBP4 to bind to Ca_v1.4, but prevent effects of CaBP4 on voltage-dependent activation and inactivation [73]. Decreased Ca_v1.4 channel availability would be expected to inhibit levels of PR glutamate release, thus reducing the dynamic range of PR signaling in these patients.

Summary

In IHCs and PRs, respectively, Ca_v1.3 and Ca_v1.4 share a number of biophysical properties that are important for their roles in mediating tonic neurotransmitter release at relatively negative membrane potentials, which is modulated by sensory stimuli. The growing number of visual and auditory disorders linked to altered Ca_v1.3 and Ca_v1.4 function emphasizes the need to better understand the roles and regulation of these channels in their native cell-types. Complicating matters is the fact that both Ca_v1.3 and Ca_v1.4 are expected to interact with a variety of proteins [91, 92]. Defining how genetic alterations in Ca_v1.3 and Ca_v1.4, and their interacting proteins, affect the localization and function of these channels in IHCs and PR remains an important challenge for understanding the pathophysiology of the associated channelopathies, as well as for the development of novel therapies.