Versatile Viral Vector Strategies for Postscreening Target Validation and RNAi ON-Target Control.

Our approach aims to optimize postscreening target validation strategies using viral vector-driven RNA interference (RNAi) cell models. The RNAiONE validation platform is an array of plasmid-based expression vectors that each drives tandem expression of the gene of interest (GOI) with one small hairpin RNA (shRNA) from a set of computed candidate sequences. The best-performing shRNA (>85% silencing efficiency) is then integrated in an inducible, all-in-one lentiviral vector to transduce pharmacologically relevant cell types that endogenously express the GOI. VariCHECK is used subsequently to combine the inducible knockdown with an equally inducible rescue of the GOI for ON-target phenotype verification. The complete RNAiONE-VariCHECK system relies on three key elements to ensure high predictability: (1) maximized silencing efficiencies by a focused shRNA validation process, (2) homogeneity of the RNAi cell pools by application of sophisticated viral vector technologies, and (3) exploiting the advantages of inducible expression systems. By using a reversible expression system, our strategy adds critical information to hot candidates from RNAi screens and avoids potential side effects that may be caused by other, irreversible genomic manipulation methods such as transcription activator-like effector nucleases (TALEN) or clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas). This approach will add credibility to top-hit screening candidates and protect researchers from costly misinterpretations early in the preclinical drug development process.

Competition between α-actinin and Ca²âº-calmodulin controls surface retention of the L-type Ca²âº channel Ca(V)1.2.

Regulation of neuronal excitability and cardiac excitation-contraction coupling requires the proper localization of L-type Ca²âº channels. We show that the actin-binding protein α-actinin binds to the C-terminal surface targeting motif of α11.2, the central pore-forming Ca(V)1.2 subunit, in order to foster its surface expression. Disruption of α-actinin function by dominant-negative or small hairpin RNA constructs reduces Ca(V)1.2 surface localization in human embryonic kidney 293 and neuronal cultures and dendritic spine localization in neurons. We demonstrate that calmodulin displaces α-actinin from their shared binding site on α11.2 upon Ca²âº influx through L-type channels, but not through NMDAR, thereby triggering loss of Ca(V)1.2 from spines. Coexpression of a Ca²âº-binding-deficient calmodulin mutant does not affect basal Ca(V)1.2 surface expression but inhibits its internalization upon Ca²âº influx. We conclude that α-actinin stabilizes Ca(V)1.2 at the plasma membrane and that its displacement by Ca²âº-calmodulin triggers Ca²âº-induced endocytosis of Ca(V)1.2, thus providing an important negative feedback mechanism for Ca²âº influx.

Calretinin regulates Ca2+-dependent inactivation and facilitation of Ca(v)2.1 Ca2+ channels through a direct interaction with the α12.1 subunit.

Voltage-gated Ca(v)2.1 Ca(2+) channels undergo dual modulation by Ca(2+), Ca(2+)-dependent inactivation (CDI), and Ca(2+)-dependent facilitation (CDF), which can influence synaptic plasticity in the nervous system. Although the molecular determinants controlling CDI and CDF have been the focus of intense research, little is known about the factors regulating these processes in neurons. Here, we show that calretinin (CR), a Ca(2+)-binding protein highly expressed in subpopulations of neurons in the brain, inhibits CDI and enhances CDF by binding directly to α(1)2.1. Screening of a phage display library with CR as bait revealed a highly basic CR-binding domain (CRB) present in multiple copies in the cytoplasmic linker between domains II and III of α(1)2.1. In pulldown assays, CR binding to fusion proteins containing these CRBs was largely Ca(2+)-dependent. α(1)2.1 coimmunoprecipitated with CR antibodies from transfected cells and mouse cerebellum, which confirmed the existence of CR-Ca(v)2.1 complexes in vitro and in vivo. In HEK293T cells, CR significantly decreased Ca(v)2.1 CDI and increased CDF. CR binding to α(1)2.1 was required for these effects, because they were not observed upon substitution of the II-III linker of α(1)2.1 with that from the Ca(v)1.2 α(1) subunit (α(1)1.2), which lacks the CRBs. In addition, coexpression of a protein containing the CRBs blocked the modulatory action of CR, most likely by competing with CR for interactions with α(1)2.1. Our findings highlight an unexpected role for CR in directly modulating effectors such as Ca(v)2.1, which may have major consequences for Ca(2+) signaling and neuronal excitability.

Distinct localization and modulation of Cav1.2 and Cav1.3 L-type Ca2+ channels in mouse sinoatrial node.

Dysregulation of L-type Ca(2+) currents in sinoatrial nodal (SAN) cells causes cardiac arrhythmia. Both Ca(v)1.2 and Ca(v)1.3 channels mediate sinoatrial L-type currents. Whether these channels exhibit differences in modulation and localization, which could affect their contribution to pacemaking, is unknown. In this study, we characterized voltage-dependent facilitation (VDF) and subcellular localization of Ca(v)1.2 and Ca(v)1.3 channels in mouse SAN cells and determined how these properties of Ca(v)1.3 affect sinoatrial pacemaking in a mathematical model. Whole cell Ba(2+) currents were recorded from SAN cells from mice carrying a point mutation that renders Ca(v)1.2 channels relatively insensitive to dihydropyridine antagonists. The Ca(v)1.2-mediated current was isolated in the presence of nimodipine (1 µm), which was subtracted from the total current to yield the Ca(v)1.3 component. With strong depolarizations (+80 mV), Ca(v)1.2 underwent significantly stronger inactivation than Ca(v)1.3. VDF of Ca(v)1.3 was evident during recovery from inactivation at a time when Ca(v)1.2 remained inactivated. By immunofluorescence, Ca(v)1.3 colocalized with ryanodine receptors in sarcomeric structures while Ca(v)1.2 was largely restricted to the delimiting plasma membrane. Ca(v)1.3 VDF enhanced recovery of pacemaker activity after pauses and positively regulated pacemaking during slow heart rate in a numerical model of mouse SAN automaticity, including preferential coupling of Ca(v)1.3 to ryanodine receptor-mediated Ca(2+) release. We conclude that strong VDF and colocalization with ryanodine receptors in mouse SAN cells are unique properties that may underlie a specific role for Ca(v)1.3 in opposing abnormal slowing of heart rate.

CaBP1 regulates voltage-dependent inactivation and activation of Ca(V)1.2 (L-type) calcium channels.

CaBP1 is a Ca(2+)-binding protein that regulates the gating of voltage-gated (Ca(V)) Ca(2+) channels. In the Ca(V)1.2 channel α(1)-subunit (α(1C)), CaBP1 interacts with cytosolic N- and C-terminal domains and blunts Ca(2+)-dependent inactivation. To clarify the role of the α(1C) N-terminal domain in CaBP1 regulation, we compared the effects of CaBP1 on two alternatively spliced variants of α(1C) containing a long or short N-terminal domain. In both isoforms, CaBP1 inhibited Ca(2+)-dependent inactivation but also caused a depolarizing shift in voltage-dependent activation and enhanced voltage-dependent inactivation (VDI). In binding assays, CaBP1 interacted with the distal third of the N-terminal domain in a Ca(2+)-independent manner. This segment is distinct from the previously identified calmodulin-binding site in the N terminus. However, deletion of a segment in the proximal N-terminal domain of both α(1C) isoforms, which spared the CaBP1-binding site, inhibited the effect of CaBP1 on VDI. This result suggests a modular organization of the α(1C) N-terminal domain, with separate determinants for CaBP1 binding and transduction of the effect on VDI. Our findings expand the diversity and mechanisms of Ca(V) channel regulation by CaBP1 and define a novel modulatory function for the initial segment of the N terminus of α(1C).

Homeostatic switch in hebbian plasticity and fear learning after sustained loss of Cav1.2 calcium channels.

Ca(2+) influx through postsynaptic Ca(v)1.x L-type voltage-gated channels (LTCCs) is particularly effective in activating neuronal biochemical signaling pathways that might be involved in Hebbian synaptic plasticity (i.e., long-term potentiation and depression) and learning and memory. Here, we demonstrate that Ca(v)1.2 is the functionally relevant LTCC isoform in the thalamus-amygdala pathway of mice. We further show that acute pharmacological block of LTCCs abolishes Hebbian plasticity in the thalamus-amygdala pathway and impairs the acquisition of conditioned fear. On the other hand, chronic genetic loss of Ca(v)1.2 triggers a homeostatic change of the synapse, leading to a fundamental alteration of the mechanism of Hebbian plasticity by synaptic incorporation of Ca(2+)-permeable, GluA2-lacking AMPA receptors. Our results demonstrate for the first time the importance of the Ca(v)1.2 LTCC subtype in synaptic plasticity and fear memory acquisition.

Ca2+-dependent facilitation of Cav1.3 Ca2+ channels by densin and Ca2+/calmodulin-dependent protein kinase II.

Ca(v)1 (L-type) channels and calmodulin-dependent protein kinase II (CaMKII) are key regulators of Ca(2+) signaling in neurons. CaMKII directly potentiates the activity of Ca(v)1.2 and Ca(v)1.3 channels, but the underlying molecular mechanisms are incompletely understood. Here, we report that the CaMKII-associated protein densin is required for Ca(2+)-dependent facilitation of Ca(v)1.3 channels. While neither CaMKII nor densin independently affects Ca(v)1.3 properties in transfected HEK293T cells, the two together augment Ca(v)1.3 Ca(2+) currents during repetitive, but not sustained, depolarizing stimuli. Facilitation requires Ca(2+), CaMKII activation, and its association with densin, as well as densin binding to the Ca(v)1.3 alpha(1) subunit C-terminal domain. Ca(v)1.3 channels and densin are targeted to dendritic spines in neurons and form a complex with CaMKII in the brain. Our results demonstrate a novel mechanism for Ca(2+)-dependent facilitation that may intensify postsynaptic Ca(2+) signals during high-frequency stimulation.

Compensatory regulation of Cav2.1 Ca2+ channels in cerebellar Purkinje neurons lacking parvalbumin and calbindin D-28k.

Ca(v)2.1 channels regulate Ca(2+) signaling and excitability of cerebellar Purkinje neurons. These channels undergo a dual feedback regulation by incoming Ca(2+) ions, Ca(2+)-dependent facilitation and inactivation. Endogenous Ca(2+)-buffering proteins, such as parvalbumin (PV) and calbindin D-28k (CB), are highly expressed in Purkinje neurons and therefore may influence Ca(v)2.1 regulation by Ca(2+). To test this, we compared Ca(v)2.1 properties in dissociated Purkinje neurons from wild-type (WT) mice and those lacking both PV and CB (PV/CB(-/-)). Unexpectedly, P-type currents in WT and PV/CB(-/-) neurons differed in a way that was inconsistent with a role of PV and CB in acute modulation of Ca(2+) feedback to Ca(v)2.1. Ca(v)2.1 currents in PV/CB(-/-) neurons exhibited increased voltage-dependent inactivation, which could be traced to decreased expression of the auxiliary Ca(v)beta(2a) subunit compared with WT neurons. Although Ca(v)2.1 channels are required for normal pacemaking of Purkinje neurons, spontaneous action potentials were not different in WT and PV/CB(-/-) neurons. Increased inactivation due to molecular switching of Ca(v)2.1 beta-subunits may preserve normal activity-dependent Ca(2+) signals in the absence of Ca(2+)-buffering proteins in PV/CB(-/-) Purkinje neurons.