Cerebellar ataxia by enhanced Ca(V)2.1 currents is alleviated by Ca2+-dependent K+-channel activators in Cacna1a(S218L) mutant mice.

Mutations in the CACNA1A gene are associated with neurological disorders, such as ataxia, hemiplegic migraine, and epilepsy. These mutations affect the pore-forming α(1A)-subunit of Ca(V)2.1 channels and thereby either decrease or increase neuronal Ca(2+) influx. A decreased Ca(V)2.1-mediated Ca(2+) influx has been shown to reduce the regularity of cerebellar Purkinje cell activity and to induce episodic cerebellar ataxia. However, little is known about how ataxia can be caused by CACNA1A mutations that increase the Ca(2+) influx, such as the S218L missense mutation. Here, we demonstrate that the S218L mutation causes a negative shift of voltage dependence of Ca(V)2.1 channels of mouse Purkinje cells and results in lowered thresholds for somatic action potentials and dendritic Ca(2+) spikes and in disrupted firing patterns. The hyperexcitability of Cacna1a(S218L) Purkinje cells was counteracted by application of the activators of Ca(2+)-dependent K(+) channels, 1-EBIO and chlorzoxazone (CHZ). Moreover, 1-EBIO also alleviated the irregularity of Purkinje cell firing both in vitro and in vivo, while CHZ improved the irregularity of Purkinje cell firing in vitro as well as the motor performance of Cacna1a(S218L) mutant mice. The current data suggest that abnormalities in Purkinje cell firing contributes to cerebellar ataxia induced by the S218L mutation and they advocate a general therapeutic approach in that targeting Ca(2+)-dependent K(+) channels may be beneficial for treating ataxia not only in patients suffering from a decreased Ca(2+) influx, but also in those suffering from an increased Ca(2+) influx in their Purkinje cells.

Ca(V)1 and Ca(V)2 channels engage distinct modes of Ca(2+) signaling to control CREB-dependent gene expression.

Activity-dependent gene expression triggered by Ca(2+) entry into neurons is critical for learning and memory, but whether specific sources of Ca(2+) act distinctly or merely supply Ca(2+) to a common pool remains uncertain. Here, we report that both signaling modes coexist and pertain to Ca(V)1 and Ca(V)2 channels, respectively, coupling membrane depolarization to CREB phosphorylation and gene expression. Ca(V)1 channels are advantaged in their voltage-dependent gating and use nanodomain Ca(2+) to drive local CaMKII aggregation and trigger communication with the nucleus. In contrast, Ca(V)2 channels must elevate [Ca(2+)](i) microns away and promote CaMKII aggregation at Ca(V)1 channels. Consequently, Ca(V)2 channels are ~10-fold less effective in signaling to the nucleus than are Ca(V)1 channels for the same bulk [Ca(2+)](i) increase. Furthermore, Ca(V)2-mediated Ca(2+) rises are preferentially curbed by uptake into the endoplasmic reticulum and mitochondria. This source-biased buffering limits the spatial spread of Ca(2+), further attenuating Ca(V)2-mediated gene expression.

Excitation-transcription coupling in sympathetic neurons and the molecular mechanism of its initiation.

In excitable cells, membrane depolarization and activation of voltage-gated Ca²+ (Ca(V)) channels trigger numerous cellular responses, including muscle contraction, secretion, and gene expression. Yet, while the mechanisms underlying excitation-contraction and excitation-secretion coupling have been extensively characterized, how neuronal activity is coupled to gene expression has remained more elusive. In this article, we will discuss recent progress toward understanding the relationship between patterns of channel activity driven by membrane depolarization and activation of the nuclear transcription factor CREB. We show that signaling strength is steeply dependent on membrane depolarization and is more sensitive to the open probability of Ca(V) channels than the Ca²+ entry itself. Furthermore, our data indicate that by decoding Ca(V) channel activity, CaMKII (a Ca²+/calmodulin-dependent protein kinase) links membrane excitation to activation of CREB in the nucleus. Together, these results revealed some interesting and unexpected similarities between excitation-transcription coupling and other forms of excitation-response coupling.

High cortical spreading depression susceptibility and migraine-associated symptoms in Ca(v)2.1 S218L mice.

OBJECTIVE: The CACNA1A gene encodes the pore-forming subunit of neuronal Ca(V)2.1 Ca2+ channels. In patients, the S218L CACNA1A mutation causes a dramatic hemiplegic migraine syndrome that is associated with ataxia, seizures, and severe, sometimes fatal, brain edema often triggered by only a mild head trauma. METHODS: We introduced the S218L mutation into the mouse Cacna1a gene and studied the mechanisms for the S218L syndrome by analyzing the phenotypic, molecular, and electrophysiological consequences. RESULTS: Cacna1a(S218L) mice faithfully mimic the associated clinical features of the human S218L syndrome. S218L neurons exhibit a gene dosage-dependent negative shift in voltage dependence of Ca(V)2.1 channel activation, resulting in enhanced neurotransmitter release at the neuromuscular junction. Cacna1a(S218L) mice also display an exquisite sensitivity to cortical spreading depression (CSD), with a vastly reduced triggering threshold, an increased propagation velocity, and frequently multiple CSD events after a single stimulus. In contrast, mice bearing the R192Q CACNA1A mutation, which in humans causes a milder form of hemiplegic migraine, typically exhibit only a single CSD event after one triggering stimulus. INTERPRETATION: The particularly low CSD threshold and the strong tendency to respond with multiple CSD events make the S218L cortex highly vulnerable to weak stimuli and may provide a mechanistic basis for the dramatic phenotype seen in S218L mice and patients. Thus, the S218L mouse model may prove a valuable tool to further elucidate mechanisms underlying migraine, seizures, ataxia, and trauma-triggered cerebral edema.

CaMKII locally encodes L-type channel activity to signal to nuclear CREB in excitation-transcription coupling.

Communication between cell surface proteins and the nucleus is integral to many cellular adaptations. In the case of ion channels in excitable cells, the dynamics of signaling to the nucleus are particularly important because the natural stimulus, surface membrane depolarization, is rapidly pulsatile. To better understand excitation-transcription coupling we characterized the dependence of cAMP response element-binding protein phosphorylation, a critical step in neuronal plasticity, on the level and duration of membrane depolarization. We find that signaling strength is steeply dependent on depolarization, with sensitivity far greater than hitherto recognized. In contrast, graded blockade of the Ca(2+) channel pore has a remarkably mild effect, although some Ca(2+) entry is absolutely required. Our data indicate that Ca(2+)/CaM-dependent protein kinase II acting near the channel couples local Ca(2+) rises to signal transduction, encoding the frequency of Ca(2+) channel openings rather than integrated Ca(2+) flux-a form of digital logic.

Spinocerebellar ataxia type 6 knockin mice develop a progressive neuronal dysfunction with age-dependent accumulation of mutant CaV2.1 channels.

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disorder caused by CAG repeat expansions within the voltage-gated calcium (Ca(V)) 2.1 channel gene. It remains controversial whether the mutation exerts neurotoxicity by changing the function of Ca(V)2.1 channel or through a gain-of-function mechanism associated with accumulation of the expanded polyglutamine protein. We generated three strains of knockin (KI) mice carrying normal, expanded, or hyperexpanded CAG repeat tracts in the Cacna1a locus. The mice expressing hyperexpanded polyglutamine (Sca6(84Q)) developed progressive motor impairment and aggregation of mutant Ca(V)2.1 channels. Electrophysiological analysis of cerebellar Purkinje cells revealed similar Ca(2+) channel current density among the three KI models. Neither voltage sensitivity of activation nor inactivation was altered in the Sca6(84Q) neurons, suggesting that expanded CAG repeat per se does not affect the intrinsic electrophysiological properties of the channels. The pathogenesis of SCA6 is apparently linked to an age-dependent process accompanied by accumulation of mutant Ca(V)2.1 channels.

The Timothy syndrome mutation differentially affects voltage- and calcium-dependent inactivation of CaV1.2 L-type calcium channels.

Calcium entry into excitable cells is an important physiological signal, supported by and highly sensitive to the activity of voltage-gated Ca2+ channels. After membrane depolarization, Ca2+ channels first open but then undergo various forms of negative feedback regulation including voltage- and calcium-dependent inactivation (VDI and CDI, respectively). Inactivation of Ca2+ channel activity is perturbed in a rare yet devastating disorder known as Timothy syndrome (TS), whose features include autism or autism spectrum disorder along with severe cardiac arrhythmia and developmental abnormalities. Most cases of TS arise from a sporadic single nucleotide change that generates a mutation (G406R) in the pore-forming subunit of the L-type Ca2+ channel Ca(V)1.2. We found that the TS mutation powerfully and selectively slows VDI while sparing or possibly speeding the kinetics of CDI. The deceleration of VDI was observed when the L-type channels were expressed with beta1 subunits prominent in brain, as well as beta2 subunits of importance for the heart. Dissociation of VDI and CDI was further substantiated by measurements of Ca2+ channel gating currents and by analysis of another channel mutation (I1624A) that hastens VDI, acting upstream of the step involving Gly406. As highlighted by the TS mutation, CDI does not proceed to completeness but levels off at approximately 50%, consistent with a change in gating modes and not an absorbing inactivation process. Thus, the TS mutation offers a unique perspective on mechanisms of inactivation as well as a promising starting point for exploring the underlying pathophysiology of autism.

Familial hemiplegic migraine.

Migraine is a severely debilitating episodic disorder affecting up to 12% of the general population. Migraine arises from both genetic and environmental factors, complicating our understanding of what makes the migraine brain susceptible to attacks. In recent years, powerful genetic screening tools have revealed several single genes linked to migraine. One example of a monogenic subtype of migraine is familial hemiplegic migraine (FHM), a rare form of migraine with aura. The fact that FHM and common multifactorial migraine have many overlapping clinical features indicates that they likely share underlying pathophysiological pathways. In addition, the identification of monogenic subtypes has made it possible to generate suitable animal models for migraine. The purpose of this review is to present an overview of the clinical features of migraine and discuss the continuing highway of migraine gene discovery. The genes associated with FHM will be discussed, including what we have learned from studying the functional consequences of FHM mutations in cellular and animal models.

The tumor suppressor eIF3e mediates calcium-dependent internalization of the L-type calcium channel CaV1.2.

Voltage-gated calcium channels (VGCCs) convert electrical activity into calcium (Ca2+) signals that regulate cellular excitability, differentiation, and connectivity. The magnitude and kinetics of Ca2+ signals depend on the number of VGCCs at the plasma membrane, but little is known about the regulation of VGCC surface expression. We report that electrical activity causes internalization of the L-type Ca2+ channel (LTC) CaV1.2 and that this is mediated by binding to the tumor suppressor eIF3e/Int6 (eukaryotic initiation factor 3 subunit e). Using total internal reflection microscopy, we identify a population of CaV1.2 containing endosomes whose rapid trafficking is strongly regulated by Ca2+. We define a domain in the II-III loop of CaV1.2 that binds eIF3e and is essential for the activity dependence of both channel internalization and endosomal trafficking. These findings provide a mechanism for activity-dependent internalization and trafficking of CaV1.2 and provide a tantalizing link between Ca2+ homeostasis and a mammalian oncogene.

L-type calcium channel ligands block nicotine-induced signaling to CREB by inhibiting nicotinic receptors.

Nicotinic acetylcholine receptors (nAChRs) are inhibited by several drugs that are commonly thought to be specific for L-type calcium channels (LTCCs). In neurons, LTCCs are activated by nicotine-induced depolarization to engage downstream signaling events; however, the role of LTCC drug interactions with nAChRs in signaling has not been examined in detail. We investigated the effects of LTCC ligands on nAChR currents and downstream signaling in rat superior cervical ganglion (SCG) neurons. We found that 10microM nicotine and 40mM K(+) both reversibly depolarize SCG neurons to -20mV, sufficient to activate LTCCs and downstream signaling, including induction of nuclear phospho-CREB (pCREB); this induction was blocked by LTCC antagonists. Interestingly, the effects of LTCC antagonists on nicotine-induced signaling to CREB are not mediated by their actions on LTCCs, but rather via inhibition of nAChRs, which prevents nicotine-induced depolarization. We show that this effect is sufficient to block pCREB induction in neurons expressing an antagonist-insensitive LTCC. Taken together, our data show that, at concentrations typically used to block LTCCs, these antagonists inhibit nAChR currents and downstream signaling. These findings serve as a caution in attributing a role for LTCCs when using these drugs experimentally or therapeutically.

Gating deficiency in a familial hemiplegic migraine type 1 mutant P/Q-type calcium channel.

Familial hemiplegic migraine type 1 (FHM1) arises from missense mutations in the gene encoding alpha1A, the pore-forming subunit of P/Q-type calcium channels. The nature of the channel disorder is fundamental to the disease, yet is not well understood. We studied how the most prevalent FHM1 mutation, a threonine to methionine substitution at position 666 (TM), affects both ionic current and gating current associated with channel activation, a previously unexplored feature of P/Q channels. Whole-cell currents were measured in HEK293 cells expressing channels containing either wild-type (WT) or TM alpha1A. Calcium currents were significantly smaller in cells expressing TM channels, consistent with previous reports. In contrast, surface expression of TM channels, measured by immunostaining against an extracellular epitope, was not decreased, and Western blots demonstrated that TM alpha1A subunits were expressed as full-length proteins. WT and TM gating currents were isolated by replacing Ca2+ with the nonpermeant cation La3+. The gating currents generated by the mutant channels were one-third that of WT, a deficiency sufficient to account for the observed attenuation in calcium current; the remaining gating current was no different in kinetics or voltage dependence. Thus, the decreased calcium influx seen with TM channels can be attributed to a reduced number of channels available to undergo the voltage-dependent conformational changes needed for channel opening, not to fewer channel proteins expressed on the cell surface. This identification of an intrinsic defect in FHM1 mutant channels helps explain their impact on neurotransmission when they occupy type-specific slots for P/Q channels at central nerve terminals.

The calcium channel ligand FPL 64176 enhances L-type but inhibits N-type neuronal calcium currents.

One strategy for isolating neuronal L-type calcium (Ca(2+)) currents, which typically comprise a minority of the whole cell current in neurons, has been to use pharmacological agents that increase channel activity. This study examines the effects of the benzoyl pyrrole FPL 64176 (FPL) on L-type Ca(2+) currents and compares them to those of the dihydropyridine (+)-202-791. At micromolar concentrations, both agonists increased whole cell current amplitude in PC12 cells. However, FPL also significantly slowed the rate of activation and elicited a longer-lasting slow component of the tail current compared to (+)-202-791. In single channel cell-attached patch recordings, FPL increased open probability, first latency, mean closed time and mean open time more than (+)-202-791, with no difference in unitary conductance. These gating differences suggest that, compared to (+)-202-791, FPL decreases transition rates between open and closed conformations. Where examined, the actions of FPL and (+)-202-791 on whole cell L-type currents in sympathetic neurons appeared similar to those in PC12 cells. In contrast to its effects on L-type current, 10 microM FPL inhibited the majority of the whole cell current in HEK cells expressing a recombinant N-type Ca(2+) channel, raising caution concerning the use of FPL as a selective L-type Ca(2+) channel agonist in neurons.