Voltage-Gated Calcium Channels: Key Players in Sensory Coding in the Retina and the Inner Ear.

Calcium influx through voltage-gated Ca (CaV) channels is the first step in synaptic transmission. This review concerns CaV channels at ribbon synapses in primary sense organs and their specialization for efficient coding of stimuli in the physical environment. Specifically, we describe molecular, biochemical, and biophysical properties of the CaV channels in sensory receptor cells of the retina, cochlea, and vestibular apparatus, and we consider how such properties might change over the course of development and contribute to synaptic plasticity. We pay particular attention to factors affecting the spatial arrangement of CaV channels at presynaptic, ribbon-type active zones, because the spatial relationship between CaV channels and release sites has been shown to affect synapse function critically in a number of systems. Finally, we review identified synaptopathies affecting sensory systems and arising from dysfunction of L-type, CaV1.3, and CaV1.4 channels or their protein modulatory elements.

A novel calcium channel Cavß2 splice variant with unique properties predominates in the retina.

Cavß subunits are essential for surface expression of voltage-gated calcium channel complexes and crucially modulate biophysical properties like voltage-dependent inactivation. Here, we describe the discovery and characterization of a novel Cavß2 variant with distinct features that predominates in the retina. We determined spliced exons in retinal transcripts of the Cacnb2 gene, coding for Cavß2, by RNA-Seq data analysis and quantitative PCR. We cloned a novel Cavß2 splice variant from mouse retina, which we are calling ß2i, and investigated biophysical properties of calcium currents with this variant in a heterologous expression system as well as its intrinsic membrane interaction when expressed alone. Our data showed that ß2i predominated in the retina with expression in photoreceptors and bipolar cells. Furthermore, we observed that the ß2i N-terminus exhibited an extraordinary concentration of hydrophobic residues, a distinct feature not seen in canonical variants. The biophysical properties resembled known membrane-associated variants, and ß2i exhibited both a strong membrane association and a propensity for clustering, which depended on hydrophobic residues in its N-terminus. We considered available Cavß structure data to elucidate potential mechanisms underlying the observed characteristics but resolved N-terminus structures were lacking and thus, precluded clear conclusions. With this description of a novel N-terminus variant of Cavß2, we expand the scope of functional variation through N-terminal splicing with a distinct form of membrane attachment. Further investigation of the molecular mechanisms underlying the features of ß2i could provide new angles on the way Cavß subunits modulate Ca2+ channels at the plasma membrane.

Cav1.4 dysfunction and congenital stationary night blindness type 2.

Cav1.4 L-type Ca2+ channels are predominantly expressed in retinal neurons, particularly at the photoreceptor terminals where they mediate sustained Ca2+ entry needed for continuous neurotransmitter release at their ribbon synapses. Cav1.4 channel gating properties are controlled by accessory subunits, associated regulatory proteins, and also alternative splicing. In humans, mutations in the CACNA1F gene encoding for Cav1.4 channels are associated with X-linked retinal disorders such as congenital stationary night blindness type 2. Mutations in the Cav1.4 protein result in a spectrum of altered functional channel activity. Several mouse models broadened our understanding of the role of Cav1.4 channels not only as Ca2+ source at retinal synapses but also as synaptic organizers. In this review, we highlight different structural and functional phenotypes of Cav1.4 mutations that might also occur in patients with congenital stationary night blindness type 2. A further important yet mostly neglected aspect that we discuss is the influence of alternative splicing on channel dysfunction. We conclude that currently available functional phenotyping strategies should be refined and summarize potential specific therapeutic options for patients carrying Cav1.4 mutations. Importantly, the development of new therapeutic approaches will permit a deeper understanding of not only the disease pathophysiology but also the physiological function of Cav1.4 channels in the retina.

The Physiology, Pathology, and Pharmacology of Voltage-Gated Calcium Channels and Their Future Therapeutic Potential.

Voltage-gated calcium channels are required for many key functions in the body. In this review, the different subtypes of voltage-gated calcium channels are described and their physiologic roles and pharmacology are outlined. We describe the current uses of drugs interacting with the different calcium channel subtypes and subunits, as well as specific areas in which there is strong potential for future drug development. Current therapeutic agents include drugs targeting L-type Ca(V)1.2 calcium channels, particularly 1,4-dihydropyridines, which are widely used in the treatment of hypertension. T-type (Ca(V)3) channels are a target of ethosuximide, widely used in absence epilepsy. The auxiliary subunit α2δ-1 is the therapeutic target of the gabapentinoid drugs, which are of value in certain epilepsies and chronic neuropathic pain. The limited use of intrathecal ziconotide, a peptide blocker of N-type (Ca(V)2.2) calcium channels, as a treatment of intractable pain, gives an indication that these channels represent excellent drug targets for various pain conditions. We describe how selectivity for different subtypes of calcium channels (e.g., Ca(V)1.2 and Ca(V)1.3 L-type channels) may be achieved in the future by exploiting differences between channel isoforms in terms of sequence and biophysical properties, variation in splicing in different target tissues, and differences in the properties of the target tissues themselves in terms of membrane potential or firing frequency. Thus, use-dependent blockers of the different isoforms could selectively block calcium channels in particular pathologies, such as nociceptive neurons in pain states or in epileptic brain circuits. Of important future potential are selective Ca(V)1.3 blockers for neuropsychiatric diseases, neuroprotection in Parkinson's disease, and resistant hypertension. In addition, selective or nonselective T-type channel blockers are considered potential therapeutic targets in epilepsy, pain, obesity, sleep, and anxiety. Use-dependent N-type calcium channel blockers are likely to be of therapeutic use in chronic pain conditions. Thus, more selective calcium channel blockers hold promise for therapeutic intervention.

Differential neuronal targeting of a new and two known calcium channel ß4 subunit splice variants correlates with their regulation of gene expression.

The ß subunits of voltage-gated calcium channels regulate surface expression and gating of CaV1 and CaV2 α1 subunits and thus contribute to neuronal excitability, neurotransmitter release, and calcium-induced gene regulation. In addition, certain ß subunits are targeted into the nucleus, where they interact directly with the epigenetic machinery. Whereas their involvement in this multitude of functions is reflected by a great molecular heterogeneity of ß isoforms derived from four genes and abundant alternative splicing, little is known about the roles of individual ß variants in specific neuronal functions. In the present study, an alternatively spliced ß4 subunit lacking the variable N terminus (ß4e) is identified. It is highly expressed in mouse cerebellum and cultured cerebellar granule cells (CGCs) and modulates P/Q-type calcium currents in tsA201 cells and CaV2.1 surface expression in neurons. Compared with the other two known full-length ß4 variants (ß4a and ß4b), ß4e is most abundantly expressed in the distal axon, but lacks nuclear-targeting properties. To determine the importance of nuclear targeting of ß4 subunits for transcriptional regulation, we performed whole-genome expression profiling of CGCs from lethargic (ß4-null) mice individually reconstituted with ß4a, ß4b, and ß4e. Notably, the number of genes regulated by each ß4 splice variant correlated with the rank order of their nuclear-targeting properties (ß4b > ß4a > ß4e). Together, these findings support isoform-specific functions of ß4 splice variants in neurons, with ß4b playing a dual role in channel modulation and gene regulation, whereas the newly detected ß4e variant serves exclusively in calcium-channel-dependent functions.

Spectrum of Cav1.4 dysfunction in congenital stationary night blindness type 2.

Defective retinal synaptic transmission in patients affected with congenital stationary night blindness type 2 (CSNB2) can result from different dysfunction phenotypes in Cav1.4 L-type calcium channels. Here we investigated two prototypical Cav1.4 variants from either end of the functional spectrum. Using whole-cell and single-channel patch-clamp techniques, we provide analysis of the biophysical characteristics of the point mutation L860P and the C-terminal truncating mutation R1827X. L860P showed a typical loss-of-function phenotype attributed to a reduced number of functional channels expressed at the plasma membrane as implied by gating current and non-stationary noise analyses. This phenotype can be rationalized, because the inserted proline is predicted to break an amphipatic helix close to the transmembrane segment IIIS1 and thus to reduce channel stability and promote misfolding. In fact, L860P was subject to an increased turnover. In contrast, R1827X displayed an apparent gain-of-function phenotype, i.e., due to a hyperpolarizing shift of the IV-curve and increased single-channel activity. However, truncation also resulted in the loss of functional C-terminal modulation and thus unmasked calcium-dependent inactivation. Thus R1827X failed to support continuous calcium influx. Current inactivation curtails the dynamic range of photoreceptors (e.g., when adapting to variation in illumination). Taken together, the analysis of two representative mutations that occur in CSNB2 patients revealed fundamental differences in the underlying defect. These may explain subtle variations in the clinical manifestation and must be taken into account, if channel function is to be restored by pharmacochaperones or related approaches.

What can naturally occurring mutations tell us about Ca(v)1.x channel function?

Voltage-gated Ca²âº channels allow for Ca²âº-dependent intracellular signaling by directly mediating Ca²âº ion influx, by physical coupling to intracellular Ca²âº release channels or functional coupling to other ion channels such as Ca²âº activated potassium channels. L-type Ca²âº channels that comprise the family of Ca(v)1 channels are expressed in many electrically excitable tissues and are characterized by their unique sensitivity to dihydropyridines. In this issue, we summarize genetic defects in L-type Ca²âº channels and analyze their role in human diseases (Ca²âº channelopathies); e.g. mutations in Ca(v)1.2 α1 cause Timothy and Brugada syndrome, mutations in Ca(v)1.3 α1 are linked to sinoatrial node dysfunction and deafness while mutations in Ca(v)1.4 α1 are associated with X-linked retinal disorders such as an incomplete form of congenital stationary night blindness. Herein, we also put the mutations underlying the channel's dysfunction into the structural context of the pore-forming α1 subunit. This analysis highlights the importance of combining functional data with structural analysis to gain a deeper understanding for the disease pathophysiology as well as for physiological channel function. This article is part of a Special Issue entitled: Calcium channels.

Characterization of two pathological gating-charge substitutions in Cav1.4 L-type calcium channels.

Cav1.4 L-type calcium channels are predominantly expressed at the photoreceptor terminals and in bipolar cells, mediating neurotransmitter release. Mutations in its gene, CACNA1F, can cause congenital stationary night-blindness type 2 (CSNB2). Due to phenotypic variability in CSNB2, characterization of pathological variants is necessary to better determine pathological mechanism at the site of action. A set of known mutations affects conserved gating charges in the S4 voltage sensor, two of which have been found in male CSNB2 patients. Here, we describe two disease-causing Cav1.4 mutations with gating charge neutralization, exchanging an arginine 964 with glycine (RG) or arginine 1288 with leucine (RL). In both, charge neutralization was associated with a reduction channel expression also reflected in smaller ON gating currents. In RL channels, the strong decrease in whole-cell current densities might additionally be explained by a reduction of single-channel currents. We further identified alterations in their biophysical properties, such as a hyperpolarizing shift of the activation threshold and an increase in slope factor of activation and inactivation. Molecular dynamic simulations in RL substituted channels indicated water wires in both, resting and active, channel states, suggesting the development of omega (ω)currents as a new pathological mechanism in CSNB2. This sum of the respective channel property alterations might add to the differential symptoms in patients beside other factors, such as genomic and environmental deviations.

Functional properties of a newly identified C-terminal splice variant of Cav1.3 L-type Ca2+ channels.

An intramolecular interaction between a distal (DCRD) and a proximal regulatory domain (PCRD) within the C terminus of long Ca(v)1.3 L-type Ca(2+) channels (Ca(v)1.3(L)) is a major determinant of their voltage- and Ca(2+)-dependent gating kinetics. Removal of these regulatory domains by alternative splicing generates Ca(v)1.3(42A) channels that activate at a more negative voltage range and exhibit more pronounced Ca(2+)-dependent inactivation. Here we describe the discovery of a novel short splice variant (Ca(v)1.3(43S)) that is expressed at high levels in the brain but not in the heart. It lacks the DCRD but, in contrast to Ca(v)1.3(42A), still contains PCRD. When expressed together with α2δ1 and ß3 subunits in tsA-201 cells, Ca(v)1.3(43S) also activated at more negative voltages like Ca(v)1.3(42A) but Ca(2+)-dependent inactivation was less pronounced. Single channel recordings revealed much higher channel open probabilities for both short splice variants as compared with Ca(v)1.3(L). The presence of the proximal C terminus in Ca(v)1.3(43S) channels preserved their modulation by distal C terminus-containing Ca(v)1.3- and Ca(v)1.2-derived C-terminal peptides. Removal of the C-terminal modulation by alternative splicing also induced a faster decay of Ca(2+) influx during electrical activities mimicking trains of neuronal action potentials. Our findings extend the spectrum of functionally diverse Ca(v)1.3 L-type channels produced by tissue-specific alternative splicing. This diversity may help to fine tune Ca(2+) channel signaling and, in the case of short variants lacking a functional C-terminal modulation, prevent excessive Ca(2+) accumulation during burst firing in neurons. This may be especially important in neurons that are affected by Ca(2+)-induced neurodegenerative processes.

Charge-Converting Nanoemulsions as Promising Retinal Drug and Gene Delivery Systems.

AIM: This study aimed to develop phosphatase-responsive ζ potential converting nanocarriers utilizing polyphosphate-coated cell-penetrating peptide (CPP)-decorated nanoemulsions (NEs) as a novel gene delivery system to retinal cells. METHODS: Poly-l-lysine (PLL) was first conjugated with oleylamine (OA) only at its carboxylic end to form the amphiphilic PLL-oleylamine (PLOA) conjugate. Afterward, NEs were loaded with PLOA prior to being coated with tripolyphosphate (TPP) to generate PLOA/TPP NEs. A plasmid containing a reporter gene for green fluorescent protein plasmid (pGFP) was complexed with cationic surfactants forming hydrophobic ion pairs that were loaded in the oily core of NEs. Phosphate removal, ζ potential conversion, and cytotoxicity of the system were evaluated. Cellular uptake and transfection efficiency were investigated in 661W photoreceptor-like cells via microscopic analysis, fluorescence spectroscopy, and flow cytometry. RESULTS: Dephosphorylation of PLOA/TPP NEs triggered by alkaline phosphatase (ALP) resulted in the exposure of positive amine groups on the surface of NE droplets and a notable conversion of the ζ potential from -22.4 to +8.5 mV. Cellular uptake of PLOA/TPP NEs performed on 661W photoreceptor-like cells showed a 3-fold increase compared to control NEs. Furthermore, PLOA/TPP NEs also showed low cytotoxicity and high transfection efficacy with ∼50% of cells transfected. CONCLUSIONS: Polyphosphate-coated CPP-decorated NEs triggered by ALP could be a promising nanosystem to efficiently deliver drugs and genetic materials to photoreceptor-like cells and other retinal cells for potential treatments of retinal diseases.

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.

Knockout of CaV1.3 L-type calcium channels in a mouse model of retinitis pigmentosa.

Retinitis Pigmentosa is a genetically heterogeneous, degenerative retinal disorder characterized by gradual dysfunction and death of photoreceptors, first rods and later cones, and progressive blindness. Studies suggested that application of L-type calcium channel blockers rescues photoreceptors in paradigms related to Ca2+ overflow. To investigate whether Cav1.3 L-type channels have protective effects in the retina, we established a new mouse model by crossing rd10, modeling autosomal-recessive RP, with Cav1.3 deficient mice (rd10/Cav1.3KO). Our immunohistochemical analyses revealed an influence of Cav1.3 channels on the degenerative process of photoreceptors. The absence of Cav1.3 delayed the centre-to-periphery degeneration of rods indicated by a significantly higher number of photoreceptor rows and, consequently, of cones. In accordance with a preserved number of cones we observed a regular row of cone somas in rd10/Cav1.3-KO retinas. Surviving rod photoreceptors maintained synaptic contacts with rod bipolar cells. However, the delay in degeneration was only observed up to postnatal day 45. Although we observed a reduction in the spontaneous oscillatory retinal activity during multielectrode array analyses, measurable functional preservation was lacking in behavioural tests. In conclusion, Cav1.3 channels contribute to photoreceptor degeneration in rd10 retinas but photoreceptor temporary rescue might rather be achieved indirectly through other retinal cell layers.

Function of cone and cone-related pathways in CaV1.4 IT mice.

CaV1.4 L-type calcium channels are predominantly expressed in photoreceptor terminals playing a crucial role for synaptic transmission and, consequently, for vision. Human mutations in the encoding gene are associated with congenital stationary night blindness type-2. Besides rod-driven scotopic vision also cone-driven photopic responses are severely affected in patients. The present study therefore examined functional and morphological changes in cones and cone-related pathways in mice carrying the CaV1.4 gain-of function mutation I756T (CaV1.4-IT) using multielectrode array, patch-clamp and immunohistochemical analyses. CaV1.4-IT ganglion cell responses to photopic stimuli were seen only in a small fraction of cells indicative of a major impairment in the cone pathway. Though cone photoreceptors underwent morphological rearrangements, they retained their ability to release glutamate. Our functional data suggested a postsynaptic cone bipolar cell defect, supported by the fact that the majority of cone bipolar cells showed sprouting, while horizontal cells maintained contacts with cones and cone-to-horizontal cell input was preserved. Furthermore a reduction of basal Ca2+ influx by a calcium channel blocker was not sufficient to rescue synaptic transmission deficits caused by the CaV1.4-IT mutation. Long term treatments with low-dose Ca2+ channel blockers might however be beneficial reducing Ca2+ toxicity without major effects on ganglion cells responses.

Channelopathies in Cav1.1, Cav1.3, and Cav1.4 voltage-gated L-type Ca2+ channels.

Voltage-gated Ca2+ channels couple membrane depolarization to Ca2+-dependent intracellular signaling events. This is achieved by mediating Ca2+ ion influx or by direct conformational coupling to intracellular Ca2+ release channels. The family of Cav1 channels, also termed L-type Ca2+ channels (LTCCs), is uniquely sensitive to organic Ca2+ channel blockers and expressed in many electrically excitable tissues. In this review, we summarize the role of LTCCs for human diseases caused by genetic Ca2+ channel defects (channelopathies). LTCC dysfunction can result from structural aberrations within their pore-forming alpha1 subunits causing hypokalemic periodic paralysis and malignant hyperthermia sensitivity (Cav1.1 alpha1), incomplete congenital stationary night blindness (CSNB2; Cav1.4 alpha1), and Timothy syndrome (Cav1.2 alpha1; reviewed separately in this issue). Cav1.3 alpha1 mutations have not been reported yet in humans, but channel loss of function would likely affect sinoatrial node function and hearing. Studies in mice revealed that LTCCs indirectly also contribute to neurological symptoms in Ca2+ channelopathies affecting non-LTCCs, such as Cav2.1 alpha1 in tottering mice. Ca2+ channelopathies provide exciting disease-related molecular detail that led to important novel insight not only into disease pathophysiology but also to mechanisms of channel function.

C-terminal modulator controls Ca2+-dependent gating of Ca(v)1.4 L-type Ca2+ channels.

Tonic neurotransmitter release at sensory cell ribbon synapses is mediated by calcium (Ca2+) influx through L-type voltage-gated Ca2+ channels. This tonic release requires the channels to inactivate slower than in other tissues. Ca(v)1.4 L-type voltage-gated Ca2+ channels (LTCCs) are found at high densities in photoreceptor terminals, and alpha1 subunit mutations cause human congenital stationary night blindness type-2 (CSNB2). Ca(v)1.4 voltage-dependent inactivation is slow and Ca2+-dependent inactivation (CDI) is absent. We show that removal of the last 55 or 122 (C122) C-terminal amino acid residues of the human alpha1 subunit restores calmodulin-dependent CDI and shifts voltage of half-maximal activation to more negative potentials. The C terminus must therefore form part of a mechanism that prevents calmodulin-dependent CDI of Ca(v)1.4 and controls voltage-dependent activation. Fluorescence resonance energy transfer experiments in living cells revealed binding of C122 to C-terminal motifs mediating CDI in other Ca2+ channels. The absence of this modulatory mechanism in the CSNB2 truncation mutant K1591X underlines its importance for normal retinal function in humans.

Assessment of the Retina of Plp-α-Syn Mice as a Model for Studying Synuclein-Dependent Diseases.

Purpose: Synucleinopathies such as multiple system atrophy (MSA) and Parkinson's disease are associated with a variety of visual symptoms. Functional and morphological retinal aberrations are therefore supposed to be valuable biomarkers for these neurodegenerative diseases. This study examined the retinal morphology and functionality resulting from human α-synuclein (α-Syn) overexpression in the transgenic Plp-α-Syn mouse model. Methods: Immunohistochemistry on retinal sections and whole-mounts was performed on 8- to 11-week-old and 12-month-old Plp-α-Syn mice and C57BL/6N controls. Quantitative RT-PCR experiments were performed to study the expression of endogenous and human α-Syn and tyrosine hydroxylase (TH). We confirmed the presence of human α-Syn in the retina in western blot analyses. Multi-electrode array (MEA) analyses from light-stimulated whole-mounted retinas were used to investigate their functionality. Results: Biochemical and immunohistochemical analyses showed human α-Syn in the retina of Plp-α-Syn mice. We found distinct staining in different retinal cell layers, most abundantly in rod bipolar cells of the peripheral retina. In the periphery, we also observed a trend toward a decline in the number of retinal ganglion cells. The number of TH+ neurons was unaffected in this human α-Syn overexpression model. MEA recordings showed that Plp-α-Syn retinas were functional but exhibited mild alterations in dim light conditions. Conclusions: Together, these findings implicate an impairment of retinal neurons in the Plp-α-Syn mouse. The phenotype partly relates to retinal deficits reported in MSA patients. We further propose the suitability of the Plp-α-Syn retina as a biological model to study synuclein-mediated mechanisms.

Expression and 1,4-dihydropyridine-binding properties of brain L-type calcium channel isoforms.

The L-type calcium channel (LTCC) isoforms Ca(v)1.2 and Ca(v)1.3 display similar 1,4-dihydropyridine (DHP) binding properties and are both expressed in mammalian brain. Recent work implicates Ca(v)1.3 channels as interesting drug targets, but no isoform-selective modulators exist. It is also unknown to what extent Ca(v)1.1 and Ca(v)1.4 contribute to L-type-specific DHP binding activity in brain. To address this question and to determine whether DHPs can discriminate between Ca(v)1.2 and Ca(v)1.3 binding pockets, we combined radioreceptor assays and quantitative polymerase chain reaction (qPCR). We bred double mutants (Ca(v)-DM) from mice expressing mutant Ca(v)1.2 channels [Ca(v)1.2DHP(-/-)] lacking high affinity for DHPs and from Ca(v)1.3 knockouts [Ca(v)1.3(-/-)]. (+)-[(3)H]isradipine binding to Ca(v)1.2DHP(-/-) and Ca(v)-DM brains was reduced to 15.1 and 4.4% of wild type, respectively, indicating that Ca(v)1.3 accounts for 10.7% of brain LTCCs. qPCR revealed that Ca(v)1.1 and Ca(v)1.4 alpha(1) subunits comprised 0.08% of the LTCC transcripts in mouse whole brain, suggesting that they cannot account for the residual binding. Instead, this could be explained by low-affinity binding (127-fold K(d) increase) to the mutated Ca(v)1.2 channels. Inhibition of (+)-[(3)H]isradipine binding to Ca(v)1.2DHP(-/-) (predominantly Ca(v)1.3) and wild-type (predominantly Ca(v)1.2) brain membranes by unlabeled DHPs revealed a 3- to 4-fold selectivity of nitrendipine and nifedipine for the Ca(v)1.2 binding pocket, a finding further confirmed with heterologously expressed channels. This suggests that small differences in their binding pockets may allow development of isoform-selective modulators for LTCCs and that, because of their very low expression, Ca(v)1.1 and Ca(v)1.4 are unlikely to serve as drug targets to treat CNS diseases.

Use-dependent block of voltage-gated Cav2.1 Ca2+ channels by petasins and eudesmol isomers.

Migraine is a frequent and often disabling disease. Treatment is unsatisfactory in many patients. A disturbed dynamic balance between excitatory and inhibitory signal processing with enhanced cortical activity probably underlies common forms of migraine. Presynaptic voltage-gated Ca(2+) channels are critical determinants of neurotransmitter release and also contribute to trigeminovascular signal transduction. Because clinical evidence exists for migraine-prophylactic actions of Petasites hybridus extracts, we investigated whether petasins comprising the main constituents of the extract inhibit currents through presynaptic Ca(v)2.1 channels expressed in Xenopus laevis oocytes. P. hybridus extract (0.02 mg/ml), petasin, neopetasin, isopetasin, S-petasin, and iso-S-petasin (50 microM) were weak tonic blockers of Ca(v)2.1-mediated barium currents (I(Ba)) during infrequent depolarizations (0.1 Hz), but their inhibitory potency increased at higher stimulation rates (1 Hz), indicating preferential block of open and/or inactivated channels. Sulfur-containing compounds (S-petasin, Iso-S-petasin) were the most potent significantly promoting the accumulation of Ca(v)2.1 channel in inactivated states during pulse trains (I(Ba) decrease during 1-Hz pulse trains: control, 45%, S-petasin, 79%; iso-S-petasin, 80%). For the Eucalyptus williamsiania sesquiterpenes alpha- and gamma-eudesmol, a comparable use-dependent inhibition was found in addition to a tonic block component. Alpha-eudesmol and petasins accelerated the voltage-dependent inactivation of Ca(v)2.1 channels during depolarizations. We demonstrate that S-petasin, iso-S-petasin, and eudesmol are Ca(v)2.1 channel inhibitors preferentially acting as use-dependent channel blockers and with the sulfur-containing substituent in position 3 of the petasins serving as important functional feature. The Ca(v)2.1-inhibitory properties of these petasins may contribute to migraine-prophylactic properties described for P. hybridus extracts.

Molecular nature of anomalous L-type calcium channels in mouse cerebellar granule cells.

Single-channel analysis revealed the existence of neuronal L-type Ca2+ channels (LTCCs) with fundamentally different gating properties; in addition to LTCCs resembling cardiac channels, LTCCs with anomalous gating were identified in a variety of neurons, including cerebellar granule cells. Anomalous LTCC gating is mainly characterized by long reopenings after repolarization following strong depolarizations or trains of action potentials. To elucidate the unknown molecular nature of anomalous LTCCs, we performed single-channel patch-clamp recordings from cerebellar granule cells of wild-type, Ca(v)1.3-/- and Ca(v)1.2DHP-/- [containing a mutation in the Ca(v)1.2 alpha1 subunit that eliminates dihydropyridine (DHP) sensitivity] mice. Quantitative reverse transcription-PCR revealed that Ca(v)1.2 accounts for 89% and Ca(v)1.3 for 11% of the LTCC transcripts in wild-type cerebellar granule cells, whereas Ca(v)1.1 and Ca(v)1.4 are expressed at insignificant levels. Anomalous LTCCs were observed in neurons of Ca(v)1.3-/- mice with a frequency not different from wild type. In the presence of the DHP agonist (+)-(S)-202-791, the typical prepulse-induced reopenings of anomalous LTCCs after repolarization were shorter in Ca(v)1.2DHP-/- neurons than in Ca(v)1.3-/- neurons. Reopenings in Ca(v)1.2DHP-/- neurons in the presence of the DHP agonist were similar to those in wild-type neurons in the absence of the agonist. These data show that Ca(v)1.2alpha1 subunits are the pore-forming subunits of anomalous LTCCs in mouse cerebellar granule cells. Given the evidence that Ca(v)1.2 channels are specifically involved in sustained Ras-MAPK (mitogen-activated protein kinase)-dependent cAMP response element-binding protein phosphorylation and LTCC-dependent hippocampal long-term potentiation (LTP) (Moosmang et al., 2005), we discuss the hypothesis that anomalous rather than cardiac-type Ca(v)1.2 channels are specifically involved in LTCC-dependent and gene transcription-dependent LTP.

Relevance of tissue specific subunit expression in channelopathies.

Channelopathies are a diverse group of human disorders that are caused by mutations in genes coding for ion channels or channel-regulating proteins. Several dozen channelopathies have been identified that involve both non-excitable cells as well as electrically active tissues like brain, skeletal and smooth muscle or the heart. In this review, we start out from the general question which ion channel genes are expressed tissue-selectively. We mined the human gene expression database Human Protein Atlas (HPA) for tissue-enriched ion channel genes and found 85 genes belonging to the ion channel families. Most of these genes were enriched in brain, testis and muscle and a complete list of the enriched ion channel genes is provided. We further focused on the tissue distribution of voltage-gated calcium channel (VGCC) genes including different brain areas and the retina based on the human gene expression from the FANTOM5 dataset. The expression data is complemented by an overview of the tissue-dependent aspects of L-type calcium channel (LTCC) function, dysfunction and pharmacology, as well as of their splice variants. Finally, we focus on the pathology of tissue-restricted LTCC channelopathies and their treatment options. This article is part of the Special Issue entitled 'Channelopathies.'