Evgeny Kobrinsky

Examining Intracellular Organelle Function Using Fluorescent Probes: From Animalcules to Quantum Dots

Fluorescence microscopy imaging has become one of the most useful techniques to assess the activity of individual cells, subcellular trafficking of signals to and between organelles, and to appreciate how organelle function is regulated. The past 2 decades have seen a tremendous advance in the rational design and development in the nature and selectivity of probes to serve as reporters of the intracellular environment in live cells. These probes range from small organic fluorescent molecules to fluorescent biomolecules and photoproteins ingeniously engineered to follow signaling traffic, sense ionic and nonionic second messengers, and report various kinase activities. These probes, together with recent advances in imaging technology, have enabled significantly enhanced spatial and temporal resolution. This review summarizes some of these developments and their applications to assess intracellular organelle function.

Differential Role of the α1C Subunit Tails in Regulation of the Cav1.2 Channel by Membrane Potential, β Subunits, and Ca2+ Ions

Voltage-gated Cav1.2 channels are composed of the pore-forming α1C and auxiliary β and α2δ subunits. Voltage-dependent conformational rearrangements of the α1C subunit C-tail have been implicated in Ca2+ signal transduction. In contrast, the α1C N-tail demonstrates limited voltage-gated mobility. We have asked whether these properties are critical for the channel function. Here we report that transient anchoring of the α1C subunit C-tail in the plasma membrane inhibits Ca2+-dependent and slow voltage-dependent inactivation. Both α2δ and β subunits remain essential for the functional channel. In contrast, if α1C subunits with are expressed α2δ but in the absence of a β subunit, plasma membrane anchoring of the α1C N terminus or its deletion inhibit both voltage- and Ca2+-dependent inactivation of the current. The following findings all corroborate the importance of the α1C N-tail/β interaction: (i) co-expression of β restores inactivation properties, (ii) release of the α1C N terminus inhibits the β-deficient channel, and (iii) voltage-gated mobility of the α1C N-tail vis à vis the plasma membrane is increased in the β-deficient (silent) channel. Together, these data argue that both the α1C N- and C-tails have important but different roles in the voltage- and Ca2+-dependent inactivation, as well as β subunit modulation of the channel. The α1C N-tail may have a role in the channel trafficking and is a target of the β subunit modulation. The β subunit facilitates voltage gating by competing with the N-tail and constraining its voltage-dependent rearrangements. Thus, cross-talk between the α1C C and N termini, β subunit, and the cytoplasmic pore region confers the multifactorial regulation of Cav1.2 channels.

Voltage-gated Mobility of the Ca2+ Channel Cytoplasmic Tails and Its Regulatory Role

Transient increase in intracellular free Ca2+ concentration generated by the voltage-gated Cav1.2 channels acts as an important intracellular signal. By using fluorescence resonance energy transfer combined with patch clamp in living cells, we present evidence for voltage-gated mobility of the cytoplasmic tails of the Cav1.2 channel and for its regulatory role in intracellular signaling. Anchoring of the C-terminal tail to the plasma membrane caused an inhibition of its state-dependent mobility, channel inactivation, and CREB-dependent transcription. Release of the tail restored these functions suggesting a direct role for voltage-gated mobility of the C-terminal tail in Ca2+ signaling.

Molecular Rearrangements of the Kv2.1 Potassium Channel Termini Associated with Voltage Gating

The voltage-gated Kv2.1 channel is composed of four identical subunits folded around the central pore and does not inactivate appreciably during short depolarizing pulses. To study voltage-induced relative molecular rearrangements of the channel, Kv2.1 subunits were genetically fused with enhanced cyan fluorescent protein and/or enhanced yellow fluorescent protein, expressed in COS1 cells, and investigated using fluorescence resonance energy transfer (FRET) microscopy combined with patch clamp. Fusion of fluorophores to either or both termini of the Kv2.1 monomer did not significantly affect the gating properties of the channel. FRET between the N- and C-terminal tags fused to the same or different Kv2.1 monomers decreased upon activation of the channel by depolarization from -80 to +60 mV, suggesting voltage-gated relative rearrangement between the termini. Because FRET between the Kv2.1 N- or C-terminal tags and the membrane-trapped EYFPN-PH pleckstrin homology domains did not change on depolarization, voltage-gated relative movements between the Kv2.1 termini occurred in a plane parallel to the plasma membrane, within a distance of 1-10 nm. FRET between the N-terminal tags did not change upon depolarization, indicating that the N termini do not rearrange relative to each other, but they could either move cooperatively with the Kv2.1 tetramer or not move at all. No FRET was detected between the C-terminal tags. Assuming their randomized orientation in the symmetrically arranged Kv2.1 subunits, C termini may move outwards in order to produce relative rearrangements between N and C termini upon depolarization.

Calmodulin-dependent gating of Cav1.2 calcium channels in the absence of Cavβ subunits

It is generally accepted that to generate calcium currents in response to depolarization, Cav1.2 calcium channels require association of the pore-forming α1C subunit with accessory Cavβ and α2δ subunits. A single calmodulin (CaM) molecule is tethered to the C-terminal α1C-LA/IQ region and mediates Ca2+-dependent inactivation of the channel. Cavβ subunits are stably associated with the α1C-interaction domain site of the cytoplasmic linker between internal repeats I and II and also interact dynamically, in a Ca2+-dependent manner, with the α1C-IQ region. Here, we describe a surprising discovery that coexpression of exogenous CaM (CaMex) with α1C/α2δ in COS1 cells in the absence of Cavβ subunits stimulates the plasma membrane targeting of α1C, facilitates calcium channel gating, and supports Ca2+-dependent inactivation. Neither real-time PCR with primers complementary to monkey Cavβ subunits nor coimmunoprecipitation analysis with exogenous α1C revealed an induction of endogenous Cavβ subunits that could be linked to the effect of CaMex. Coexpression of a calcium-insensitive CaM mutant CaM1234 also facilitated gating of Cavβ-free Cav1.2 channels but did not support Ca2+-dependent inactivation. Our results show there is a functional matchup between CaMex and Cavβ subunits that, in the absence of Cavβ, renders Ca2+ channel gating facilitated by CaM molecules other than the one tethered to LA/IQ to support Ca2+-dependent inactivation. Thus, coexpression of CaMex creates conditions when the channel gating, voltage- and Ca2+-dependent inactivation, and plasma-membrane targeting occur in the absence of Cavβ. We suggest that CaMex affects specific Cavβ-free conformations of the channel that are not available to endogenous CaM.

New Determinant for the CaVβ2 Subunit Modulation of the CaV1.2 Calcium Channel

Cavβ subunits support voltage gating of Cav1.2 calcium channels and play important role in excitation-contraction coupling. The common central membrane-associated guanylate kinase (MAGUK) region of Cavβ binds to the α-interaction domain (AID) and the IQ motif of the pore-forming α1C subunit, but these two interactions do not explain why the cardiac Cavβ2 subunit splice variants differentially modulate inactivation of Ca2+ currents (ICa). Previously we described β2Δg, a functionally active splice variant of human Cavβ2 lacking MAGUK. By deletion analysis of β2Δg, we have now identified a 41-amino acid C-terminal essential determinant (β2CED) that stimulates ICa in the absence of Cavβ subunits and conveys a +20-mV shift in the peak of the ICa-voltage relationship. The β2CED is targeted by α1C to the plasma membrane, forms a complex with α1C but does not bind to AID. Electrophysiology and binding studies point to the calmodulin-interacting LA/IQ region in the α1C subunit C terminus as a functionally relevant β2CED binding site. The β2CED interacts with LA/IQ in a Ca2+- and calmodulin-independent manner and need LA, but not IQ, to activate the channel. Deletion/mutation analyses indicated that each of the three Cavβ2/α1C interactions is sufficient to support ICa. However, β2CED does not support Ca2+-dependent inactivation, suggesting that interactions of MAGUK with AID and IQ are crucial for Ca2+-induced inactivation. The β2CED is conserved only in Cavβ2 subunits. Thus, β2CED constitutes a previously unknown integrative part of the multifactorial mechanism of Cavβ2-subunit differential modulation of the Cav1.2 calcium channel that in β2Δg occurs without MAGUK.

Oligomerization of Cavβ subunits is an essential correlate of Ca2+ channel activity

Voltage-gated calcium channels conduct Ca(2+) ions in response to membrane depolarization. The resulting transient increase in cytoplasmic free calcium concentration is a critical trigger for the initiation of such vital responses as muscle contraction and transcription. L-type Ca(v)1.2 calcium channels are complexes of the pore-forming α(1C) subunit associated with cytosolic Ca(v)β subunits. All major Ca(v)βs share a highly homologous membrane associated guanylate kinase-like (MAGUK) domain that binds to α(1C) at the α-interaction domain (AID), a short motif in the linker between transmembrane repeats I and II. In this study we show that Ca(v)β subunits form multimolecular homo- and heterooligomeric complexes in human vascular smooth muscle cells expressing native calcium channels and in Cos7 cells expressing recombinant Ca(v)1.2 channel subunits. Ca(v)βs oligomerize at the α(1C) subunits residing in the plasma membrane and bind to the AID. However, Ca(v)β oligomerization occurs independently on the association with α(1C). Molecular structures responsible for Ca(v)β oligomerization reside in 3 regions of the guanylate kinase subdomain of MAGUK. An augmentation of Ca(v)β homooligomerization significantly increases the calcium current density, while heterooligomerization may also change the voltage-dependence and inactivation kinetics of the channel. Thus, oligomerization of Ca(v)β subunits represents a novel and essential aspect of calcium channel regulation.

Effect of Cavβ Subunits on Structural Organization of Cav1.2 Calcium Channels

Background Voltage-gated Cav1.2 calcium channels play a crucial role in Ca2+ signaling. The pore-forming α1C subunit is regulated by accessory Cavβ subunits, cytoplasmic proteins of various size encoded by four different genes (Cavβ1 - β4) and expressed in a tissue-specific manner. Methods and Results Here we investigated the effect of three major Cavβ types, β1b, β2d and β3, on the structure of Cav1.2 in the plasma membrane of live cells. Total internal reflection fluorescence microscopy showed that the tendency of Cav1.2 to form clusters depends on the type of the Cavβ subunit present. The highest density of Cav1.2 clusters in the plasma membrane and the smallest cluster size were observed with neuronal/cardiac β1b present. Cav1.2 channels containing β3, the predominant Cavβ subunit of vascular smooth muscle cells, were organized in a significantly smaller number of larger clusters. The inter- and intramolecular distances between α1C and Cavβ in the plasma membrane of live cells were measured by three-color FRET microscopy. The results confirm that the proximity of Cav1.2 channels in the plasma membrane depends on the Cavβ type. The presence of different Cavβ subunits does not result in significant differences in the intramolecular distance between the termini of α1C, but significantly affects the distance between the termini of neighbor α1C subunits, which varies from 67 Å with β1b to 79 Å with β3. Conclusions Thus, our results show that the structural organization of Cav1.2 channels in the plasma membrane depends on the type of Cavβ subunits present.

Microdomain organization and frequency-dependence of CREB-dependent transcriptional signaling in heart cells

Voltage‐gated Cav1.2 calcium channels couple membrane depolarization to cAMP response‐element‐binding protein (CREB)‐dependent transcriptional activation. To investigate the spatial and temporal organization of CREB‐dependent transcriptional nuclear microdomains, we combined perforated patch‐clamp technique and FRET microscopy for monitoring CREB and CREB‐binding protein interaction in the nuclei of live cells. The experimental approach to the quantitative assessment of CREB‐dependent transcriptional signaling evoked by cAMP‐ and Cav1.2‐dependent mechanisms was devised in COS1 cells expressing recombinant Cav1.2 calcium channels. Using continuous 2‐dimensional wavelet transform and time series analyses, we found that nuclear CREB‐dependent tran‐scriptional signaling is organized differentially in spatially and temporally separated microdomains of 4 distinct types. In rat neonatal cardiomyocytes, CREB‐dependent transcription is mediated by the cAMP‐initiated CaMKII‐sensitive and Cav1.2‐initiated CaMKII‐insensitive mechanisms. The latter microdomains show a tendency to exhibit periodic behavior correlated with spontaneous contraction of myocytes suggestive of frequency‐dependent CREB‐dependent transcriptional regulation in the heart.—Kobrinsky, E., Duong, S.Q., Sheydina, A., Soldatov, N. M. Microdomain organization and frequency‐dependence of CREB‐dependent transcriptional signaling in heart cells. FASEB J. 25, 1544–1555 (2011). www.fasebj.org

Ca2 +/calmodulin-activated phosphodiesterase 1A is highly expressed in rabbit cardiac sinoatrial nodal cells and regulates pacemaker function

Constitutive Ca(2+)/calmodulin (CaM)-activation of adenylyl cyclases (ACs) types 1 and 8 in sinoatrial nodal cells (SANC) generates cAMP within lipid-raft-rich microdomains to initiate cAMP-protein kinase A (PKA) signaling, that regulates basal state rhythmic action potential firing of these cells. Mounting evidence in other cell types points to a balance between Ca(2+)-activated counteracting enzymes, ACs and phosphodiesterases (PDEs) within these cells. We hypothesized that the expression and activity of Ca(2+)/CaM-activated PDE Type 1A is higher in SANC than in other cardiac cell types. We found that PDE1A protein expression was 5-fold higher in sinoatrial nodal tissue than in left ventricle, and its mRNA expression was 12-fold greater in the corresponding isolated cells. PDE1 activity (nimodipine-sensitive) accounted for 39% of the total PDE activity in SANC lysates, compared to only 4% in left ventricular cardiomyocytes (LVC). Additionally, total PDE activity in SANC lysates was lowest (10%) in lipid-raft-rich and highest (76%) in lipid-raft-poor fractions (equilibrium sedimentation on a sucrose density gradient). In intact cells PDE1A immunolabeling was not localized to the cell surface membrane (structured illumination microscopy imaging), but located approximately within about 150nm inside of immunolabeling of hyperpolarization-activated cyclic nucleotide-gated potassium channels (HCN4), which reside within lipid-raft-rich microenvironments. In permeabilized SANC, in which surface membrane ion channels are not functional, nimodipine increased spontaneous SR Ca(2+) cycling. PDE1A mRNA silencing in HL-1 cells increased the spontaneous beating rate, reduced the cAMP, and increased cGMP levels in response to IBMX, a broad spectrum PDE inhibitor (detected via fluorescence resonance energy transfer microscopy). We conclude that signaling via cAMP generated by Ca(2+)/CaM-activated AC in SANC lipid raft domains is limited by cAMP degradation by Ca(2+)/CaM-activated PDE1A in non-lipid raft domains. This suggests that local gradients of [Ca(2+)]-CaM or different AC and PDE1A affinity regulate both cAMP production and its degradation, and this balance determines the intensity of Ca(2+)-AC-cAMP-PKA signaling that drives SANC pacemaker function.