CaV1/CaV2 channels, comprised of pore-forming α1 and auxiliary (β,α2δ) subunits, control diverse biological responses in excitable cells. Molecules blocking CaV1/CaV2 channel currents (I Ca) profoundly regulate physiology and have many therapeutic applications. Rad/Rem/Rem2/Gem GTPases (RGKs) strongly inhibit CaV1/CaV2 channels. Understanding how RGKs block I Ca is critical for insights into their physiological function, and may provide design principles for developing novel CaV1/CaV2 channel inhibitors. The RGK binding sites within CaV1/CaV2 channel complexes responsible for I Ca inhibition are ambiguous, and it is unclear whether there are mechanistic differences among distinct RGKs. All RGKs bind β subunits, but it is unknown if and how this interaction contributes to I Ca inhibition. We investigated the role of RGK/β interaction in Rem inhibition of recombinant CaV1.2 channels, using a mutated β (β2aTM) selectively lacking RGK binding. Rem blocked β2aTM-reconstituted channels (74% inhibition) less potently than channels containing wild-type β2a (96% inhibition), suggesting the prevalence of both β-binding-dependent and independent modes of inhibition. Two mechanistic signatures of Rem inhibition of CaV1.2 channels (decreased channel surface density and open probability), but not a third (reduced maximal gating charge), depended on Rem binding to β. We identified a novel Rem binding site in CaV1.2 α1C N-terminus that mediated β-binding-independent inhibition. The CaV2.2 α1B subunit lacks the Rem binding site in the N-terminus and displays a solely β-binding-dependent form of channel inhibition. Finally, we discovered an unexpected functional dichotomy amongst distinct RGKs— while Rem and Rad use both β-binding-dependent and independent mechanisms, Gem and Rem2 use only a β-binding-dependent method to inhibit CaV1.2 channels. The results provide new mechanistic perspectives, and reveal unexpected variations in determinants, underlying inhibition of CaV1.2/CaV2.2 channels by distinct RGK GTPases.
The cardiac voltage-gated sodium channel (NaV1.5) underlies impulse conduction in the heart and its depolarization-induced inactivation is essential in control of the duration of the QT interval of the electrocardiogram (ECG). Perturbation of Nav1.5 inactivation by drugs or inherited mutation can underlie and trigger cardiac arrhythmias. The carboxy terminus plays an important role in channel inactivation, but complete structural information on its predicted structural domain is unknown. Here we measure interactions between the functionally critical distal C-T alpha helix (H6) and the proximal structured EF hand motif using transition metal ion FRET. We measure distances at three loci along H6 relative to an intrinsic tryptophan, demonstrating the proximal-distal interaction in a contiguous carboxy terminus polypeptide. Using these data together with the existing NaV1.5 carboxy terminus NMR structure, we construct a model of the predicted structured region of the carboxy terminus. An arrhythmia associated H6 mutant which impairs inactivation decreases FRET, indicating destabilization of the distal-proximal intramolecular interaction. These data provide a structural correlate to the pathological phenotype of the mutant channel.
SignificanceInflux of calcium ions through surface membrane calcium channels that open in response to electrical signals is important for vital biological processes including generation of the heartbeat and nerve cell communication. Blocking such calcium channels in a tissue- and isoform-specific manner is a sought-after treatment strategy for diseases including chronic pain and Parkinson’s disease. Proteins that can be expressed in cells to selectively block different calcium channel types have particular advantages over conventional small-molecule blockers. A four-member family of proteins known as RGK proteins strongly inhibit calcium channels, but do so in a non-selective manner, limiting their potential usefulness. Here we identified mutated RGK proteins that perform as isoform-selective calcium channel blockers, advancing the therapeutic potential of these proteins.
Many metabotropic receptors in the nervous system act through signaling pathways that result in the inhibition of voltage-dependent calcium channels. Our previous findings showed that activation of seven-transmembrane receptors results in the internalization of calcium channels. This internalization takes place within a few seconds, raising the question of whether the endocytic machinery is in close proximity to the calcium channel to cause such rapid internalization. Here we show that voltage-dependent calcium channels are pre-associated with arrestin, a protein known to play a role in receptor trafficking. Upon GABA B receptor activation, receptors are recruited to the arrestin-channel complex and internalized. -Arrestin 1 selectively binds to the SNAREbinding region of the calcium channel. Peptides containing the arrestin-binding site of the channel disrupt agonist-induced channel internalization. Taken together these data suggest a novel neuronal role for arrestin.Inhibition of voltage-dependent calcium channels by seventransmembrane receptors (7TMR) 2 is one of the primary means of regulation of calcium-dependent physiological processes such as synaptic transmission, muscle contraction, and membrane excitability. In neurons, the Ca v 2.2 (N-type) channel is a prominent target for G protein-mediated modulation (1, 2). Inhibition of Ca v 2.2 channels can be voltage-dependent, and mediated by direct interactions with G protein -␥ subunits (3, 4). In addition, kinases such as protein kinase C and tyrosine kinases have been shown to inhibit Ca v 2.2 channels in a voltage-independent manner (5, 6). Additional mechanisms may exist by which Ca 2ϩ influx is regulated. Dunlap and Fischbach (7) have suggested that transmitter-mediated shortening of the duration of the action potential could be due to a decrease in the number of voltage-dependent calcium channels at the membrane. Recently we have reported an additional mechanism by which 7TMRs can regulate neuronal calcium levels that involves a rapid internalization of voltage-dependent calcium channels into clathrin-coated vesicles upon receptor activation (8). Here we demonstrate that -arrestin 1 is associated with Ca v 2.2 channels and that activation of 7TMRs results in the formation of an arrestin-receptor-channel complex. This interaction is required for internalization of calcium channels and plays a role in the modulation of calcium current. EXPERIMENTAL PROCEDURESMaterials-The following primary antibodies were used in these studies: rabbit anti-pan-␣ 1 (1:200,1.5 g/ml) (Alomone Labs, Jerusalem, Israel), anti-arrestin (1:500, BD Biosciences), and anti-GABAR1 (1:200, Chemicon). Anti--arrestin 1 and anti--arrestin 2 antibodies, and recombinant -arrestin 1 and 2 (29) were kindly provided by the Lefkowitz laboratory. The following secondary antibodies were used in our studies: Oregon Green 488-conjugated goat anti-rabbit IgG (HϩL) (1:200, 10 g/ml), Cy3-conjugated goat anti-mouse IgG (HϩL) (1:200, 7.5 g/ml), and Cy5-goat anti-guinea pig IgG (HϩL) (1:200, 7.5 ...
Rad/Rem/Rem2/Gem (RGK) proteins are Ras-like GTPases that potently inhibit all high-voltage-gated calcium (CaV1/CaV2) channels and are, thus, well-positioned to tune diverse physiological processes. Understanding how RGK proteins inhibit CaV channels is important for perspectives on their (patho)physiological roles and could advance their development and use as genetically-encoded CaV channel blockers. We previously reported that Rem can block surface CaV1.2 channels in 2 independent ways that engage distinct components of the channel complex: (1) by binding auxiliary β subunits (β-binding-dependent inhibition, or BBD); and (2) by binding the pore-forming α1C subunit N-terminus (α1C-binding-dependent inhibition, or ABD). By contrast, Gem uses only the BBD mechanism to block CaV1.2. Rem molecular determinants required for BBD CaV1.2 inhibition are the distal C-terminus and the guanine nucleotide binding G-domain which interact with the plasma membrane and CaVβ, respectively. However, Rem determinants for ABD CaV1.2 inhibition are unknown. Here, combining fluorescence resonance energy transfer, electrophysiology, systematic truncations, and Rem/Gem chimeras we found that the same Rem distal C-terminus and G-domain also mediate ABD CaV1.2 inhibition, but with different interaction partners. Rem distal C-terminus interacts with α1C N-terminus to anchor the G-domain which likely interacts with an as-yet-unidentified site. In contrast to some previous studies, neither the C-terminus of Rem nor Gem was sufficient to inhibit CaV1/CaV2 channels. The results reveal that similar molecular determinants on Rem are repurposed to initiate 2 independent mechanisms of CaV1.2 inhibition.
Voltage-dependent calcium channels (VDCCs) play a pivotal role in normal excitation-contraction coupling in cardiac myocytes. These channels can be modulated through activation of -adrenergic receptors (-ARs), which leads to an increase in calcium current (I Ca-L ) density through cardiac Ca v 1 channels as a result of phosphorylation by cAMP-dependent protein kinase A. Changes in I Ca-L density and kinetics in heart failure often occur in the absence of changes in Ca v 1 channel expression, arguing for the importance of posttranslational modification of these channels in heart disease. The precise molecular mechanisms that govern the regulation of VDCCs and their cell surface localization remain unknown. Our data show that sustained -AR activation induces internalization of a cardiac macromolecular complex involving VDCC and -arrestin 1 (-Arr1) into clathrincoated vesicles. Pretreatment of myocytes with pertussis toxin prevents the internalization of VDCCs, suggesting that G i/o mediates this response. A peptide that selectively disrupts the interaction between Ca V 1.2 and -Arr1 and tyrosine kinase inhibitors readily prevent agonist-induced VDCC internalization. These observations suggest that VDCC trafficking is mediated by G protein switching to G i of the -AR, which plays a prominent role in various cardiac pathologies associated with a hyperadrenergic state, such as hypertrophy and heart failure. Regulation of voltage-dependent calcium channels (VDCCs)3 plays a pivotal role in excitation-contraction coupling in cardiac myocytes. During the action potential upstroke, membrane depolarization causes the opening of VDCCs, encoded by the pore-forming ␣ 1 subunit, Ca v 1.2 (1). Ca 2ϩ entry through VDCCs triggers the release of Ca 2ϩ from the sarcoplasmic reticulum via ryanodine receptors. Although the regulation of VDCCs in the heart has been extensively studied, key molecular mechanisms underlying channel function, trafficking, membrane targeting, retention, and internalization remain unknown. Activation of the -AR, a G protein-coupled receptor (GPCR), leads to positive inotropic effects mediated by phosphorylation of the VDCC via cAMP-dependent protein kinase A (2). This, however, is a transient phenomenon since persistent activation of the receptor causes its subsequent phosphorylation by GPCR kinases (GRKs) (3), causing the -AR to become a target for arrestin (4), which mediates the recruitment of the receptor into clathrin-coated vesicles (5).In addition to decreasing single channel permeability, persistent membrane depolarization can regulate the number of Ca v 1.2 channels at the plasma membrane. For example, sustained KCl-induced depolarization of rat cortical neurons effectively decreases Ca v 1.2 channel activity (6). Ca v 1.2 channels have been proposed to contain a membrane-targeting domain within their calmodulin (CaM)-binding domain in the C terminus (7). Pitt and colleagues (8) showed that Ca 2ϩ -CaM interaction with this domain accelerated the rate of trafficking of Ca v 1.2 channels to ...
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