Annalisa Zuccotti

Cav1.3 and BK Channels for Timing and Regulating Cell Firing

L-type Ca2+ channels (LTCCs, Cav1) open readily during membrane depolarization and allow Ca2+ to enter the cell. In this way, LTCCs regulate cell excitability and trigger a variety of Ca2+-dependent physiological processes such as: excitation–contraction coupling in muscle cells, gene expression, synaptic plasticity, neuronal differentiation, hormone secretion, and pacemaker activity in heart, neurons, and endocrine cells. Among the two major isoforms of LTCCs expressed in excitable tissues (Cav1.2 and Cav1.3), Cav1.3 appears suitable for supporting a pacemaker current in spontaneously firing cells. It has steep voltage dependence and low threshold of activation and inactivates slowly. Using Cav1.3−/− KO mice and membrane current recording techniques such as the dynamic and the action potential clamp, it has been possible to resolve the time course of Cav1.3 pacemaker currents that regulate the spontaneous firing of dopaminergic neurons and adrenal chromaffin cells. In several cell types, Cav1.3 is selectively coupled to BK channels within membrane nanodomains and controls both the firing frequency and the action potential repolarization phase. Here we review the most critical aspects of Cav1.3 channel gating and its coupling to large conductance BK channels recently discovered in spontaneously firing neurons and neuroendocrine cells with the aim of furnishing a converging view of the role that these two channel types play in the regulation of cell excitability.

The reduced cochlear output and the failure to adapt the central auditory response causes tinnitus in noise exposed rats.

Tinnitus is proposed to be caused by decreased central input from the cochlea, followed by increased spontaneous and evoked subcortical activity that is interpreted as compensation for increased responsiveness of central auditory circuits. We compared equally noise exposed rats separated into groups with and without tinnitus for differences in brain responsiveness relative to the degree of deafferentation in the periphery. We analyzed (1) the number of CtBP2/RIBEYE-positive particles in ribbon synapses of the inner hair cell (IHC) as a measure for deafferentation; (2) the fine structure of the amplitudes of auditory brainstem responses (ABR) reflecting differences in sound responses following decreased auditory nerve activity and (3) the expression of the activity-regulated gene Arc in the auditory cortex (AC) to identify long-lasting central activity following sensory deprivation. Following moderate trauma, 30% of animals exhibited tinnitus, similar to the tinnitus prevalence among hearing impaired humans. Although both tinnitus and no-tinnitus animals exhibited a reduced ABR wave I amplitude (generated by primary auditory nerve fibers), IHCs ribbon loss and high-frequency hearing impairment was more severe in tinnitus animals, associated with significantly reduced amplitudes of the more centrally generated wave IV and V and less intense staining of Arc mRNA and protein in the AC. The observed severe IHCs ribbon loss, the minimal restoration of ABR wave size, and reduced cortical Arc expression suggest that tinnitus is linked to a failure to adapt central circuits to reduced cochlear input.

Noise-Induced Inner Hair Cell Ribbon Loss Disturbs Central Arc Mobilization: A Novel Molecular Paradigm for Understanding Tinnitus

Increasing evidence shows that hearing loss is a risk factor for tinnitus and hyperacusis. Although both often coincide, a causal relationship between tinnitus and hyperacusis has not been shown. Currently, tinnitus and hyperacusis are assumed to be caused by elevated responsiveness in subcortical circuits. We examined both the impact of different degrees of cochlear damage and the influence of stress priming on tinnitus induction. We used (1) a behavioral animal model for tinnitus designed to minimize stress, (2) ribbon synapses in inner hair cells (IHCs) as a measure for deafferentation, (3) the integrity of auditory brainstem responses (ABR) to detect differences in stimulus-evoked neuronal activity, (4) the expression of the activity-regulated cytoskeletal protein, Arc, to identify long-lasting changes in network activity within the basolateral amygdala (BLA), hippocampal CA1, and auditory cortex (AC), and (5) stress priming to investigate the influence of corticosteroid on trauma-induced brain responses. We observed that IHC ribbon loss (deafferentation) leads to tinnitus when ABR functions remain reduced and Arc is not mobilized in the hippocampal CA1 and AC. If, however, ABR waves are functionally restored and Arc is mobilized, tinnitus does not occur. Both central response patterns were found to be independent of a profound threshold loss and could be shifted by the corticosterone level at the time of trauma. We, therefore, discuss the findings in the context of a history of stress that can trigger either an adaptive or nonadaptive brain response following injury.

Structural and functional differences between L-type calcium channels: crucial issues for future selective targeting

Within the family of voltage-gated calcium channels (VGCCs), L-type channels (L-VGCCs) represent a well-established therapeutic target for calcium channel blockers, which are widely used to treat hypertension and myocardial ischemia. L-VGCCs outside the cardiovascular system also control key physiological processes such as neuronal plasticity, sensory cell function (e.g. in the inner ear and retina) and endocrine function (e.g. in pancreatic beta cells and adrenal chromaffin cells). Research into L-VGCCs was stimulated by the discovery that the known L-VGCC isoforms (Ca(V)1.1, Ca(V)1.2, Ca(V)1.3 and Ca(V)1.4) possess different biophysical properties. However, no L-VGCC-isoform-selective drugs have yet been identified. In this review, we examine Ca(V)1.2 and Ca(V)1.3 isoforms at the level of genetic structure, splice variants, post-translational modifications and functional protein coupling. We discuss candidate Ca(V)1.2- and Ca(V)1.3-specific characteristics as future therapeutic targets in individual organs.

Modulation of Cav1.3 Ca2+ channel gating by Rab3 interacting molecule

Neurotransmitter release and spontaneous action potentials during cochlear inner hair cell (IHC) development depend on the activity of Ca(v)1.3 voltage-gated L-type Ca(2+) channels. Their voltage- and Ca(2+)-dependent inactivation kinetics are slower than in other tissues but the underlying molecular mechanisms are not yet understood. We found that Rab3-interacting molecule-2alpha (RIM2alpha) mRNA is expressed in immature cochlear IHCs and the protein co-localizes with Ca(v)1.3 in the same presynaptic compartment of IHCs. Expression of RIM proteins in tsA-201 cells revealed binding to the beta-subunit of the channel complex and RIM-induced slowing of both Ca(2+)- and voltage-dependent inactivation of Ca(v)1.3 channels. By inhibiting inactivation, RIM induced a non-inactivating current component typical for IHC Ca(v)1.3 currents which should allow these channels to carry a substantial window current during prolonged depolarizations. These data suggest that RIM2 contributes to the stabilization of Ca(v)1.3 gating kinetics in immature IHCs.

Lack of brain-derived neurotrophic factor hampers inner hair cell synapse physiology, but protects against noise-induced hearing loss

The precision of sound information transmitted to the brain depends on the transfer characteristics of the inner hair cell (IHC) ribbon synapse and its multiple contacting auditory fibers. We found that brain derived neurotrophic factor (BDNF) differentially influences IHC characteristics in the intact and injured cochlea. Using conditional knock-out mice (BDNFPax2 KO) we found that resting membrane potentials, membrane capacitance and resting linear leak conductance of adult BDNFPax2 KO IHCs showed a normal maturation. Likewise, in BDNFPax2 KO membrane capacitance (ΔCm) as a function of inward calcium current (ICa) follows the linear relationship typical for normal adult IHCs. In contrast the maximal ΔCm, but not the maximal size of the calcium current, was significantly reduced by 45% in basal but not in apical cochlear turns in BDNFPax2 KO IHCs. Maximal ΔCm correlated with a loss of IHC ribbons in these cochlear turns and a reduced activity of the auditory nerve (auditory brainstem response wave I). Remarkably, a noise-induced loss of IHC ribbons, followed by reduced activity of the auditory nerve and reduced centrally generated wave II and III observed in control mice, was prevented in equally noise-exposed BDNFPax2 KO mice. Data suggest that BDNF expressed in the cochlea is essential for maintenance of adult IHC transmitter release sites and that BDNF upholds opposing afferents in high-frequency turns and scales them down following noise exposure.

Ca(V)1.3-Driven SK Channel Activation Regulates Pacemaking and Spike Frequency Adaptation in Mouse Chromaffin Cells

Mouse chromaffin cells (MCCs) fire spontaneous action potentials (APs) at rest. Cav1.3 L-type calcium channels sustain the pacemaker current, and their loss results in depolarized resting potentials (Vrest), spike broadening, and remarkable switches into depolarization block after BayK 8644 application. A functional coupling between Cav1.3 and BK channels has been reported but cannot fully account for the aforementioned observations. Here, using Cav1.3−/− mice, we investigated the role of Cav1.3 on SK channel activation and how this functional coupling affects the firing patterns induced by sustained current injections. MCCs express SK1–3 channels whose tonic currents are responsible for the slow irregular firing observed at rest. Percentage of frequency increase induced by apamin was found inversely correlated to basal firing frequency. Upon stimulation, MCCs build-up Cav1.3-dependent SK currents during the interspike intervals that lead to a notable degree of spike frequency adaptation (SFA). The major contribution of Cav1.3 to the subthreshold Ca2+ charge during an AP-train rather than a specific molecular coupling to SK channels accounts for the reduced SFA of Cav1.3−/− MCCs. Low adaptation ratios due to reduced SK activation associated with Cav1.3 deficiency prevent the efficient recovery of NaV channels from inactivation. This promotes a rapid decline of AP amplitudes and facilitates early onset of depolarization block following prolonged stimulation. Thus, besides serving as pacemaker, Cav1.3 slows down MCC firing by activating SK channels that maintain NaV channel availability high enough to preserve stable AP waveforms, even upon high-frequency stimulation of chromaffin cells during stress responses.

Retrocochlear function of the peripheral deafness gene Cacna1d

Hearing impairment represents the most common sensory deficit in humans. Genetic mutations contribute significantly to this disorder. Mostly, only malfunction of the ear is considered. Here, we assessed the role of the peripheral deafness gene Cacna1d, encoding the L-type channel Ca(v)1.3, in downstream processing of acoustic information. To this end, we generated a mouse conditional Cacna1d-eGFP(flex) allele. Upon pairing with Egr2::Cre mice, Ca(v)1.3 was ablated in the auditory brainstem, leaving the inner ear intact. Structural assessment of the superior olivary complex (SOC), an essential auditory brainstem center, revealed a dramatic volume reduction (43-47%) of major nuclei in young adult Egr2::Cre;Cacna1d-eGFP(flex) mice. This volume decline was mainly caused by a reduced cell number (decline by 46-56%). Abnormal formation of the lateral superior olive was already present at P4, demonstrating an essential perinatal role of Ca(v)1.3 in the SOC. Measurements of auditory brainstem responses demonstrated a decreased amplitude in the auditory nerve between 50 and 75 dB stimulation in Egr2::Cre;Cacna1d-eGFP(flex) knockout mice and increased amplitudes in central auditory processing centers. Immunohistochemical studies linked the amplitude changes in the central auditory system to reduced expression of K(v)1.2. No changes were observed for K(v)1.1, KCC2, a determinant of inhibitory neurotransmission, and choline acetyltransferase, a marker of efferent olivocochlear neurons. Together, these analyses identify a crucial retrocochlear role of Ca(v)1.3 and demonstrate that mutations in deafness genes can affect sensory cells and neurons alike. As a corollary, hearing aids have to address central auditory processing deficits as well.

α2δ3 Is Essential for Normal Structure and Function of Auditory Nerve Synapses and Is a Novel Candidate for Auditory Processing Disorders

The auxiliary subunit α2δ3 modulates the expression and function of voltage-gated calcium channels. Here we show that α2δ3 mRNA is expressed in spiral ganglion neurons and auditory brainstem nuclei and that the protein is required for normal acoustic responses. Genetic deletion of α2δ3 led to impaired auditory processing, with reduced acoustic startle and distorted auditory brainstem responses. α2δ3−/− mice learned to discriminate pure tones, but they failed to discriminate temporally structured amplitude-modulated tones. Light and electron microscopy analyses revealed reduced levels of presynaptic Ca2+ channels and smaller auditory nerve fiber terminals contacting cochlear nucleus bushy cells. Juxtacellular in vivo recordings of sound-evoked activity in α2δ3−/− mice demonstrated impaired transmission at these synapses. Together, our results identify a novel role for the α2δ3 auxiliary subunit in the structure and function of specific synapses in the mammalian auditory pathway and in auditory processing disorders.

Equal sensitivity of Cav1.2 and Cav1.3 channels to the opposing modulations of PKA and PKG in mouse chromaffin cells

•  Cav1.2 and Cav1.3 L‐type calcium channels are highly expressed in rat and mouse chromaffin cells. Beside shaping and pacemaking action potential trains, they regulate vesicle exocytosis and endocytosis. •  L‐type channels are opposingly regulated by the cAMP–PKA and cGMP–PKG pathways and their Ca2+ current can undergo marked up and down changes. To date, most of the reported findings on L‐type channel modulation derive from the cardiac Cav1.2 isoform. •  Here, using wild‐type and Cav1.3 knock out (KO) mouse chromaffin cells we show that, like Cav1.2, Cav1.3 channels are effectively modulated by PKA and PKG at basal conditions and during maximal PKA/PKG stimulation. The extent of modulation is nearly equal for both Cav1 channel isoforms. •  PKA and PKG pathways act independently on Cav1.2 and Cav1.3, producing cumulative effects that are mostly visible when activating PKA and inhibiting PKG, or vice versa. Under these conditions the L‐type Ca2+ current can undergo changes of one order of magnitude. •  These extreme Cav1 channel modulations are likely to occur during different physiological conditions of the adrenal gland: ‘fight‐or‐flight’ response vs. relaxed states.