Vladimir Tsemakhovich

CaBP1 regulates voltage dependent inactivation and activation of Cav1.2 (L-type) calcium channels.

CaBP1 is a Ca2+-binding protein that regulates the gating of voltage-gated (CaV) Ca2+ channels. In the CaV1.2 channel α1-subunit (α1C), CaBP1 interacts with cytosolic N- and C-terminal domains and blunts Ca2+-dependent inactivation. To clarify the role of the α1C N-terminal domain in CaBP1 regulation, we compared the effects of CaBP1 on two alternatively spliced variants of α1C containing a long or short N-terminal domain. In both isoforms, CaBP1 inhibited Ca2+-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 Ca2+-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 α1C isoforms, which spared the CaBP1-binding site, inhibited the effect of CaBP1 on VDI. This result suggests a modular organization of the α1C N-terminal domain, with separate determinants for CaBP1 binding and transduction of the effect on VDI. Our findings expand the diversity and mechanisms of CaV channel regulation by CaBP1 and define a novel modulatory function for the initial segment of the N terminus of α1C.

Two distinct aspects of coupling between Gα(i) protein and G protein-activated K+ channel (GIRK) revealed by fluorescently labeled Gα(i3) protein subunits.

G protein-activated K+ channels (Kir3 or GIRK) are activated by direct interaction with Gβγ. Gα is essential for specific signaling and regulates basal activity of GIRK (Ibasal) and kinetics of the response elicited by activation by G protein-coupled receptors (Ievoked). These regulations are believed to occur within a GIRK-Gα-Gβγ signaling complex. Fluorescent energy resonance transfer (FRET) studies showed strong GIRK-Gβγ interactions but yielded controversial results regarding the GIRK-Gαi/o interaction. We investigated the mechanisms of regulation of GIRK by Gαi/o using wild-type Gαi3 (Gαi3WT) and Gαi3 labeled at three different positions with fluorescent proteins, CFP or YFP (xFP). Gαi3xFP proteins bound the cytosolic domain of GIRK1 and interacted with Gβγ in a guanine nucleotide-dependent manner. However, only an N-terminally labeled, myristoylated Gαi3xFP (Gαi3NT) closely mimicked all aspects of Gαi3WT regulation except for a weaker regulation of Ibasal. Gαi3 labeled with YFP within the Gα helical domain preserved regulation of Ibasal but failed to restore fast Ievoked. Titrated expression of Gαi3NT and Gαi3WT confirmed that regulation of Ibasal and of the kinetics of Ievoked of GIRK1/2 are independent functions of Gαi. FRET and direct biochemical measurements indicated much stronger interaction between GIRK1 and Gβγ than between GIRK1 and Gαi3. Thus, Gαi/oβγ heterotrimer may be attached to GIRK primarily via Gβγ within the signaling complex. Our findings support the notion that Gαi/o actively regulates GIRK. Although regulation of Ibasal is a function of GαiGDP, our new findings indicate that regulation of kinetics of Ievoked is mediated by GαiGTP.

Dual regulation of G proteins and the G-protein–activated K+ channels by lithium

Significance Recent genome-wide association studies suggest a strong linkage between psychiatric disorders, especially bipolar disorder (BPD) and schizophrenia, and ion channels, including G-protein–activated K+ channels (GIRK); however, there are no clear functional links. Lithium is a prominent antibipolar treatment. We report a dual regulation of GIRK channels by therapeutic doses of lithium in neurons. We reproduced these regulations in Xenopus oocytes, identified the molecular mechanism (a dual regulation of G proteins via actions on Gα subunit), and verified the action of Li+ on Gα–Gβγ interaction by direct biochemical studies. The discovery of a significant regulation of an ion channel by therapeutic doses of lithium may help linking between neuronal excitability and mechanisms of BPD. Lithium (Li+) is widely used to treat bipolar disorder (BPD). Cellular targets of Li+, such as glycogen synthase kinase 3β (GSK3β) and G proteins, have long been implicated in BPD etiology; however, recent genetic studies link BPD to other proteins, particularly ion channels. Li+ affects neuronal excitability, but the underlying mechanisms and the relevance to putative BPD targets are unknown. We discovered a dual regulation of G protein-gated K+ (GIRK) channels by Li+, and identified the underlying molecular mechanisms. In hippocampal neurons, therapeutic doses of Li+ (1–2 mM) increased GIRK basal current (Ibasal) but attenuated neurotransmitter-evoked GIRK currents (Ievoked) mediated by Gi/o-coupled G-protein–coupled receptors (GPCRs). Molecular mechanisms of these regulations were studied with heterologously expressed GIRK1/2. In excised membrane patches, Li+ increased Ibasal but reduced GPCR-induced GIRK currents. Both regulations were membrane-delimited and G protein-dependent, requiring both Gα and Gβγ subunits. Li+ did not impair direct activation of GIRK channels by Gβγ, suggesting that inhibition of Ievoked results from an action of Li+ on Gα, probably through inhibition of GTP–GDP exchange. In direct binding studies, Li+ promoted GPCR-independent dissociation of GαiGDP from Gβγ by a Mg2+-independent mechanism. This previously unknown Li+ action on G proteins explains the second effect of Li+, the enhancement of GIRKs Ibasal. The dual effect of Li+ on GIRK may profoundly regulate the inhibitory effects of neurotransmitters acting via GIRK channels. Our findings link between Li+, neuronal excitability, and both cellular and genetic targets of BPD: GPCRs, G proteins, and ion channels.

Stargazin Modulates Neuronal Voltage-dependent Ca2+ Channel Cav2.2 by a Gβγ-dependent Mechanism

Loss of neuronal protein stargazin (γ2) is associated with recurrent epileptic seizures and ataxia in mice. Initially, due to homology to the skeletal muscle calcium channel γ1 subunit, stargazin and other family members (γ3–8) were classified as γ subunits of neuronal voltage-gated calcium channels (such as CaV2.1-CaV2.3). Here, we report that stargazin interferes with G protein modulation of CaV2.2 (N-type) channels expressed in Xenopus oocytes. Stargazin counteracted the Gβγ-induced inhibition of CaV2.2 channel currents, caused either by coexpression of the Gβγ dimer or by activation of a G protein-coupled receptor. Expression of high doses of Gβγ overcame the effects of stargazin. High affinity Gβγ scavenger proteins m-cβARK and m-phosducin produced effects similar to stargazin. The effects of stargazin and m-cβARK were not additive, suggesting a common mechanism of action, and generally independent of the presence of the CaVβ3 subunit. However, in some cases, coexpression of CaVβ3 blunted the modulation by stargazin. Finally, the Gβγ-opposing action of stargazin was not unique to CaV2.2, as stargazin also inhibited the Gβγ-mediated activation of the G protein-activated K+ channel. Purified cytosolic C-terminal part of stargazin bound Gβγ in vitro. Our results suggest that the regulation by stargazin of biophysical properties of CaV2.2 are not exerted by direct modulation of the channel but via a Gβγ-dependent mechanism.

Oxidation of low-density lipoprotein by hemoglobin-hemichrome

Hemoglobin and myoglobin are inducers of low-density lipoprotein oxidation in the presence of H(2)O(2). The reaction of these hemoproteins with H(2)O(2) result in a mixture of protein products known as hemichromes. The oxygen-binding hemoproteins function as peroxidases but as compared to classic heme-peroxidases have a much lower activity on small sized and a higher one on large sized substrates. A heme-globin covalent adduct, a component identified in myoglobin-hemichrome, was reported to be the cause of myoglobin peroxidase activity on low-density lipoprotein. In this study, we analyzed the function of hemoglobin-hemichrome in low-density lipoprotein oxidation. Oxidation of lipids was analyzed by formation of conjugated diene and malondialdehyde; and oxidation of Apo-B protein was analyzed by development of bityrosine fluorescence and covalently cross-linked protein. Hemoglobin-hemichrome has indeed triggered oxidation of both lipids and protein, but unlike myoglobin, hemichrome has required the presence of H(2)O(2). In correlation to this, we found that unlike myoglobin, hemichrome formed by hemoglobin/H(2)O(2) does not contain a globin-heme covalent adduct. Nevertheless, hemoglobin-hemichrome remains oxidatively active towards LDL, indicating that other components of the oxidatively denatured hemoglobin should be considered responsible for its hazardous activity in vascular pathology.

Recruitment of Gβγ controls the basal activity of G‐protein coupled inwardly rectifying potassium (GIRK) channels: crucial role of distal C terminus of GIRK1

The G‐protein coupled inwardly rectifying potassium (GIRK) channel is an important mediator of neurotransmission via Gβγ subunit of the heterotrimeric Gi/o protein released by G‐protein coupled receptor (GPCR) activation. Channels containing the GIRK1 subunit exhibit high basal currents, whereas channels that are formed by the GIRK2 subunit have very low basal currents. GIRK1‐containing channels, but not channels consisting of GIRK2 only, recruit Gβγ to the plasma membrane. The Gα subunit of the G protein is not recruited by either GIRK1/2 or GIRK2. The unique distal C terminus of GIRK1 (G1‐dCT) endows the channel with strong interaction with Gβγ, and deletion of G1‐dCT abolishes the Gβγ recruitment and reduces the basal currents. These findings suggest that the basal activity of GIRK channels depends on channel‐induced recruitment of Gβγ. The unique C terminus of GIRK1 subunit plays an important role in Gβγ recruitment.