María A. Gandini

Functional Coupling of Rab3-interacting Molecule 1 (RIM1) and L-type Ca2+ Channels in Insulin Release

Insulin release by pancreatic β-cells is regulated by diverse intracellular signals, including changes in Ca2+ concentration resulting from Ca2+ entry through voltage-gated (CaV) channels. It has been reported that the Rab3 effector RIM1 acts as a functional link between neuronal CaV channels and the machinery for exocytosis. Here, we investigated whether RIM1 regulates recombinant and native L-type CaV channels (that play a key role in hormone secretion) and whether this regulation affects insulin release. Whole-cell patch clamp currents were recorded from HEK-293 and insulinoma RIN-m5F cells. RIM1 and CaV channel expression was identified by RT-PCR and Western blot. RIM1-CaV channel interaction was determined by co-immunoprecipitation. Knockdown of RIM1 and CaV channel subunit expression were performed using small interference RNAs. Insulin release was assessed by ELISA. Co-expression of CaV1.2 and CaV1.3 L-type channels with RIM1 in HEK-293 cells revealed that RIM1 may not determine the availability of L-type CaV channels but decreases the rate of inactivation of the whole cell currents. Co-immunoprecipitation experiments showed association of the CaVβ auxiliary subunit with RIM1. The lack of CaVβ expression suppressed channel regulation by RIM1. Similar to the heterologous system, an increase of current inactivation was observed upon knockdown of endogenous RIM1. Co-immunoprecipitation showed association of CaVβ and RIM1 in insulin-secreting RIN-m5F cells. Knockdown of RIM1 notably impaired high K+-stimulated insulin secretion in the RIN-m5F cells. These data unveil a novel functional coupling between RIM1 and the L-type CaV channels via the CaVβ auxiliary subunit that contribute to determine insulin secretion.

Constitutive and ghrelin-dependent GHSR1a activation impairs CaV2.1 and CaV2.2 currents in hypothalamic neurons

Constitutive and ligand-dependent GHSR1a activity attenuates CaV2 current and hypothalamic GABA release through distinct mechanisms and signaling pathways.

Familial hemiplegic migraine type 1 mutations W1684R and V1696I alter G protein-mediated regulation of CaV2.1 voltage-gated calcium channels

Familial hemiplegic migraine type 1 (FHM-1) is a monogenic form of migraine with aura that is characterized by recurrent attacks of a typical migraine headache with transient hemiparesis during the aura phase. In a subset of patients, additional symptoms such as epilepsy and cerebellar ataxia are part of the clinical phenotype. FHM-1 is caused by missense mutations in the CACNA1A gene that encodes the pore-forming subunit of Ca(V)2.1 voltage-gated Ca(2+) channels. Although the functional effects of an increasing number of FHM-1 mutations have been characterized, knowledge on the influence of most of these mutations on G protein regulation of channel function is lacking. Here, we explored the effects of G protein-dependent modulation on mutations W1684R and V1696I which cause FHM-1 with and without cerebellar ataxia, respectively. Both mutations were introduced into the human Ca(V)2.1α(1) subunit and their functional consequences investigated after heterologous expression in human embryonic kidney 293 (HEK-293) cells using patch-clamp recordings. When co-expressed along with the human μ-opioid receptor, application of the agonist [d-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) inhibited currents through both wild-type (WT) and mutant Ca(V)2.1 channels, which is consistent with the known modulation of these channels by G protein-coupled receptors. Prepulse facilitation, which is a way to characterize the relief of direct voltage-dependent G protein regulation, was reduced by both FHM-1 mutations. Moreover, the kinetic analysis of the onset and decay of facilitation showed that the W1684R and V1696I mutations affect the apparent dissociation and reassociation rates of the Gβγ dimer from the channel complex, suggesting that the G protein-Ca(2+) channel affinity may be altered by the mutations. These biophysical studies may shed new light on the pathophysiology underlying FHM-1.

CaV2.2 channel cell surface expression is regulated by the light chain 1 (LC1) of the microtubule-associated protein B (MAP1B) via UBE2L3-mediated ubiquitination and degradation.

Microtubule-associated protein B is a cytoskeleton protein consisting of heavy and light (LC) chains that play important roles in the regulation of neuronal morphogenesis and function. LC1 is also well known to interact with diverse ionotropic receptors at postsynapse. Much less is known, however, regarding the role of LC1 at presynaptic level where voltage-gated N-type Ca2+ channels couple membrane depolarization to neurotransmitter release. Here, we investigated whether LC1 interacts with the N-type channels. Co-localization analysis revealed spatial proximity of the two proteins in hippocampal neurons. The interaction between LC1 and the N-type channel was demonstrated using co-immunoprecipitation experiments and in vitro pull-down assays. Detailed biochemical analysis suggested that the interaction occurs through the N-terminal of LC1 and the C-terminal of the pore-forming CaVα1 subunit of the channels. Patch-clamp studies in HEK-293 cells revealed a significant decrease in N-type currents upon LC1 expression, without apparent changes in kinetics. Recordings performed in the presence of MG132 prevented the actions of LC1 suggesting enhanced channel proteasomal degradation. Interestingly, using the yeast two-hybrid system and immunoprecipitation assays in HEK-293 cells, we revealed an interaction between LC1 and the ubiquitin-conjugating enzyme UBE2L3. Furthermore, we found that the LC1/UBE2L3 complex could interact with the N-type channels, suggesting that LC1 may act as a scaffold protein to increase UBE2L3-mediated channel ubiquitination. Together these results revealed a novel functional coupling between LC1 and the N-type channels.

Functional interactions between voltage-gated Ca2 + channels and Rab3-interacting molecules (RIMs): New insights into stimulus–secretion coupling

Stimulus-secretion coupling is a complex set of intracellular reactions initiated by an external stimulus that result in the release of hormones and neurotransmitters. Under physiological conditions this signaling process takes a few milliseconds, and to minimize delays cells have developed a formidable integrated network, in which the relevant molecules are tightly packed on the nanometer scale. Active zones, the sites of release, are composed of several different proteins including voltage-gated Ca(2+) (Ca(V)) channels. It is well acknowledged that hormone and neurotransmitter release is initiated by the activation of these channels located close to docked vesicles, though the mechanisms that enrich channels at release sites are largely unknown. Interestingly, Rab3 binding proteins (RIMs), a diverse multidomain family of proteins that operate as effectors of the small G protein Rab3 involved in secretory vesicle trafficking, have recently identified as binding partners of Ca(V) channels, placing both proteins in the center of an interaction network in the molecular anatomy of the active zones that influence different aspects of secretion. Here, we review recent evidences providing support for the notion that RIMs directly bind to the pore-forming and auxiliary β subunits of Ca(V) channels and with RIM-binding protein, another interactor of the channels. Through these interactions, RIMs regulate the biophysical properties of the channels and their anchoring relative to active zones, significantly influencing hormone and neurotransmitter release.

Whole-cell patch-clamp recordings of Ca2+ currents from isolated neonatal mouse dorsal root ganglion (DRG) neurons.

Primary culture of sensory neurons from dorsal root ganglia (DRGs) is a widely used model for studying Ca(2+) channels. DRG neurons can be collected from neonate or adult mice; the production of cultures can take a couple of hours, and cells so derived can be used almost immediately or maintained for as long as 1 wk. This method allows the isolation of neurons for numerous experimental purposes, including whole-cell patch-clamp recording. The purpose of this protocol is to provide a description of methods commonly used for the harvest and growth of DRG neonatal neurons as well as for recording whole-cell currents through voltage-sensitive Ca(2+) channels in these cells.

Whole-Cell Patch-Clamp Recording of Recombinant Voltage-Sensitive Ca2+ Channels Heterologously Expressed in HEK-293 Cells

Several factors, including showing a high efficiency of transfection and protein production, faithful translation and processing of proteins, and possession of a small cell size, have together made human embryonic kidney 293 (HEK-293) cells a popular choice among electrophysiologists. This cell line has been extensively used to study recombinant heterooligomeric voltage-sensitive Ca(2+) channels. The main reasons for this are that the properties of the channel complexes can be studied in isolation and that it is also possible to define the subunit composition of the expressed channels. This protocol provides a detailed method for the transfection of, and obtaining electrophysiological data from, HEK-293 cells transiently expressing neuronal (N-type) recombinant Ca(2+) channels.

The familial hemiplegic migraine type 1 mutation K1336E affects direct G protein-mediated regulation of neuronal P/Q-type Ca2+ channels

Background Familial hemiplegic migraine type 1 (FHM-1) is an autosomal dominant form of migraine with aura characterized by recurrent migraine, hemiparesis and ataxia. FHM-1 has been linked to missense mutations in the CACNA1A gene encoding the pore-forming subunit of the neuronal voltage-gated P/Q-type Ca2+ channel (CaV2.1α1). Methods Here, we explored the effects of the FHM-1 K1336E mutation on G protein-dependent modulation of the recombinant P/Q-type channel. The mutation was introduced into the human CaV2.1α1 subunit and its functional consequences investigated after heterologous expression in HEK-293 cells using patch-clamp recordings. Results Functional analysis of the K1336E mutation revealed a reduction of Ca2+ current densities, a ∼10 mV left-shift in the current-voltage relationship, and the slowing of current inactivation kinetics. When co-expressed along with the human μ-opioid receptor, application of the agonist DAMGO inhibited whole-cell currents through both the wild-type and the mutant channels. Prepulse facilitation was also reduced by the K1336E mutation. Likewise, the kinetic analysis of the onset and decay of facilitation showed that the mutation affects the apparent dissociation and reassociation rates of the Gβγ dimer from the channel complex. Conclusions These results suggest that the extent of G-protein-mediated inhibition is significantly reduced in the K1336E mutant CaV2.1 Ca2+ channels. This alteration would contribute to render the neuronal network hyperexcitable, possibly as a consequence of reduced presynaptic inhibition, and may help to explain some aspects of the FHM-1 pathophysiology.

Reduced Hyperpolarization-Activated Current Contributes to Enhanced Intrinsic Excitability in Cultured Hippocampal Neurons from PrP(-/-) Mice.

Genetic ablation of cellular prion protein (PrPC) has been linked to increased neuronal excitability and synaptic activity in the hippocampus. We have previously shown that synaptic activity in hippocampi of PrP-null mice is increased due to enhanced N-methyl-D-aspartate receptor (NMDAR) function. Here, we focused on the effect of PRNP gene knock-out (KO) on intrinsic neuronal excitability, and in particular, the underlying ionic mechanism in hippocampal neurons cultured from P0 mouse pups. We found that the absence of PrPC profoundly affected the firing properties of cultured hippocampal neurons in the presence of synaptic blockers. The membrane impedance was greater in PrP-null neurons, and this difference was abolished by the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker ZD7288 (100 μM). HCN channel activity appeared to be functionally regulated by PrPC. The amplitude of voltage sag, a characteristic of activating HCN channel current (Ih), was decreased in null mice. Moreover, Ih peak current was reduced, along with a hyperpolarizing shift in activation gating and slower kinetics. However, neither HCN1 nor HCN2 formed a biochemical complex with PrPC. These results suggest that the absence of PrP downregulates the activity of HCN channels through activation of a cell signaling pathway rather than through direct interactions. This in turn contributes to an increase in membrane impedance to potentiate neuronal excitability.

Toxins Targeting Voltage-Activated Ca 2+ Channels and their Potential Biomedical Applications

Voltage-gated Ca(2+) (CaV) channels are transmembrane proteins primarily formed by an ion-conducting α 1 subunit that can associate with auxiliary β and α2δ subunits. Ca(2+) entering the cell through these channels serves as a versatile second messenger of electrical signaling, initiating numerous different cellular processes ranging from gene expression to cell fertilization, neuronal transmission and cell death. CaV channels, as other ion channels, are targets for numerous ligands including naturally occurring peptide toxins. Some of these peptide toxins are invaluable tools for studying their structure and function and have potential therapeutic applications. Here, we present an overview of the current knowledge regarding the structure and function of CaV channels as well as their role in human disease, and highlight some of the growing applications of peptide toxins targeting CaV channels. Analysis and understanding of the molecular strategy used by these peptide toxins might allow the design of novel classes of therapeutic agents acting on specific targets with high selectivity and efficacy.