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 ...
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