Biomedical subjects

Federica Bertaso

Publications and source records attributed to Federica Bertaso.

6 recordsLinked to original sources

PubMed2006

MacMARCKS interacts with the metabotropic glutamate receptor type 7 and modulates G protein-mediated constitutive inhibition of calcium channels.

We have previously shown that the interaction of Ca2+/calmodulin with the metabotropic glutamate receptor type 7 (mGluR7) promotes the G-protein-mediated inhibition of voltage-sensitive Ca2+ channels (VSCCs) seen upon agonist activation. Here, we performed a yeast two-hybrid screen of a new-born rat brain cDNA library using the cytoplasmic C-terminal tail of mGluR7 as bait and identified macrophage myristoylated alanine-rich c-kinase substrate (MacMARCKS) as a binding protein. The interaction was confirmed in vitro and in vivo by pull-down assays, immunoprecipitation, and colocalization of mGluR7 and MacMARCKS in transfected HEK293 cells and cultured cerebellar granule cells. Binding of MacMARCKS to mGluR7 was antagonized by Ca2+/calmodulin. In neurons, cotransfection of MacMARCKS with mGluR7, but not mGluR7 mutants unable to bind MacMARCKS, reduced the G-protein-mediated tonic inhibition of VSCCs in the absence of mGluR7 agonist. These results suggest that competitive interactions of Ca2+/calmodulin and MacMARCKS with mGluR7 control the tonic inhibition of VSCCs by G-proteins.

Amino Acid Sequence↗

PubMed2004

Dominant-negative calcium channel suppression by truncated constructs involves a kinase implicated in the unfolded protein response.

Expression of the calcium channel Ca(V)2.2 is markedly suppressed by coexpression with truncated constructs of Ca(V)2.2. Furthermore, a two-domain construct of Ca(V)2.1 mimicking an episodic ataxia-2 mutation strongly inhibited Ca(V)2.1 currents. We have now determined the specificity of this effect, identified a potential mechanism, and have shown that such constructs also inhibit endogenous calcium currents when transfected into neuronal cell lines. Suppression of calcium channel expression requires interaction between truncated and full-length channels, because there is inter-subfamily specificity. Although there is marked cross-suppression within the Ca(V)2 calcium channel family, there is no cross-suppression between Ca(V)2 and Ca(V)3 channels. The mechanism involves activation of a component of the unfolded protein response, the endoplasmic reticulum resident RNA-dependent kinase (PERK), because it is inhibited by expression of dominant-negative constructs of this kinase. Activation of PERK has been shown previously to cause translational arrest, which has the potential to result in a generalized effect on protein synthesis. In agreement with this, coexpression of the truncated domain I of Ca(V)2.2, together with full-length Ca(V)2.2, reduced the level not only of Ca(V)2.2 protein but also the coexpressed alpha2delta-2. Thapsigargin, which globally activates the unfolded protein response, very markedly suppressed Ca(V)2.2 currents and also reduced the expression level of both Ca(V)2.2 and alpha2delta-2 protein. We propose that voltage-gated calcium channels represent a class of difficult-to-fold transmembrane proteins, in this case misfolding is induced by interaction with a truncated cognate Ca(V) channel. This may represent a mechanism of pathology in episodic ataxia-2.

Animals↗

PubMed2003

Mechanism of action of Gq to inhibit G beta gamma modulation of CaV2.2 calcium channels: probed by the use of receptor-G alpha tandems.

The stable interaction of a G-protein coupled receptor and a particular partner G-protein was made possible by creating tandems between the alpha(2A) adrenergic receptor (alpha(2A)-R) and pertussis toxin-resistant mutants of different G alpha subunits of heterotrimeric G-proteins. Both alpha(2A)-R-G alpha(o) and alpha(2A)-R-G alpha(i) proved able to reconstitute agonist-induced voltage-dependent inhibition of N-type calcium channels (Ca(V)2.2) similar to the wild-type alpha(2A)-R when expressed in COS-7 cells. The interaction of G(q) with the G(i/o) signaling pathways was studied by expressing either G alpha(q) or a chimeric construct based on G alpha(q) containing the last five amino acids of G alpha(z), which is activated by alpha(2A)-R. It was found that G alpha(qz5) activated by the wild-type alpha(2A)-R inhibited Ca(V)2.2 currents in a voltage-independent fashion. Furthermore, G alpha(qz5) counteracted the voltage-dependent inhibition resulting from alpha(2A)-R-G alpha(o) activation. We subsequently investigated the basis for the behavior of G alpha(qz5). Our evidence suggests that this occurs as a result of a downstream effect of activation of G alpha(qz5) because it was blocked by C-terminal construct of phospholipase C beta 1. Furthermore it is likely to occur in part via protein kinase C (PKC) activation, because the PKC activator phorbol dibutyrate mimicked the effects of G alpha(qz5) in alpha(2A)-R-G alpha(o)-transfected cells. Conversely, cells expressing both alpha(2A)-R-G alpha(o) and G alpha(qz5) exhibited a partial restoration of voltage-dependent inhibition in the presence of the PKC inhibitor bisindolylmaleimide I (GF 109203X). The potential sites of phosphorylation are discussed.

Animals↗

PubMed2002

The novel product of a five-exon stargazin-related gene abolishes Ca(V)2.2 calcium channel expression.

We have cloned and characterized a new member of the voltage-dependent Ca(2+) channel gamma subunit family, with a novel gene structure and striking properties. Unlike the genes of other potential gamma subunits identified by their homology to the stargazin gene, CACNG7 is a five-, and not four-exon gene whose mRNA encodes a protein we have designated gamma(7). Expression of human gamma(7) has been localized specifically to brain. N-type current through Ca(V)2.2 channels was almost abolished when co-expressed transiently with gamma(7) in either Xenopus oocytes or COS-7 cells. Furthermore, immunocytochemistry and western blots show that gamma(7) has this effect by causing a large reduction in expression of Ca(V)2.2 rather than by interfering with trafficking or biophysical properties of the channel. No effect of transiently expressed gamma(7) was observed on pre-existing endogenous N-type calcium channels in sympathetic neurones. Low homology to the stargazin-like gamma subunits, different gene structure and the unique functional properties of gamma(7) imply that it represents a distinct subdivision of the family of proteins identified by their structural and sequence homology to stargazin.

Amino Acid Sequence↗

PubMed2002

Expression of voltage-gated K+ channels in human atrium.

Voltage-gated K(+) channels underlie repolarisation of the cardiac action potential and represent a potential therapeutic target in the treatment of cardiac dysrhythmias. However, very little is known about the relative expression of K(+) channel subunits in the human myocardium. We used a semi-quantitative RT-PCR technique to examine the relative expression of mRNAs for the voltage-gated K(+) channel subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, Kv4.2, Kv4.3, KvLQT1, HERG and IsK in samples of human atrial appendage. Data were expressed as a percentage expression density relative to an 18S ribosomal RNA internal standard. The most abundant K(+) channel mRNAs were Kv4.3 (80.7 +/- 10.1 %), Kv1.5 (69.7 +/- 11.2 %) and HERG (55.9 +/- 21.5 %). Significant expression of KvLQT1 (33.5 +/- 5.5 %,) and Kv1.4 (26.7 +/- 9.6 %) was also detected. Levels of mRNAs for Kv1.2 and IsK were very low and neither Kv2.1 nor Kv4.2 mRNA were detected in any experiments. Whole-cell patch-clamp techniques were used to examine the outward currents of isolated human atrial myocytes at 37 degrees C. These recordings demonstrated the existence of transient (I(to1)) and sustained (I(so)) outward currents in isolated human atrial myocytes. I(to1), and not I(so), showed voltage-dependent inactivation during 100 ms pre-pulses. Both I(to1) and I(so) were inhibited by high concentrations (2 mM) of the K(+) channel blocker, 4-aminopyridine (4-AP). However, lower concentrations of 4-AP (10 microM) inhibited I(so) selectively. I(to1) recovered from inactivation relatively rapidly (tao approximately 21 ms). These data, with published information regarding the properties of expressed K(+) channels, suggest that Kv4.3 represents the predominant K(+) channel subunit underlying I(to1) with little contribution of Kv1.4. The sensitivity of Iso to very low concentrations of 4-aminopyridine and the relatively low expression of mRNA for Kv1.2 and Kv2.1 is consistent with the major contribution of Kv1.5 to this current. The physiological significance of the expression of KvLQT1 and Kv1.4 mRNA in the human atrium warrants further investigation.

4-Aminopyridine↗

PubMed2000

An altered repolarizing potassium current in rat cardiac myocytes after subtotal nephrectomy.

Renal failure in humans is associated with electrocardiographic changes including altered QT interval dispersion, which suggests that cardiac myocyte repolarization is abnormal and which appears to correlate with cardiac prognosis. In this study, cardiac myocyte repolarizing currents have been studied in isolated cells from rats 8 wk after subtotal nephrectomy (SNx), using sham-operated animals as controls. In addition, monophasic cardiac action potentials were recorded from the epicardial surface of the left ventricle (LV) apex, LV base, and the right ventricle of isolated perfused hearts paced at 320/min. SNx was associated with cardiac hypertrophy and histologic evidence of myocardial fibrosis, but SNx rats were not hypertensive. Repolarizing K(+) currents were measured using whole-cell patch-clamp, and 4-aminopyridine (4-AP)-sensitive transient outward (I(to)) and 4-AP-insensitive sustained outward (I(so)) components were quantified. After SNx, I(to) was increased by two to threefold at voltages from -30 to +60 mV and showed increased heterogeneity. For example, at 0 mV voltage clamp pulse, the median I(to) was increased from 3.23 pA/pF in control myocytes (interquartile range 3.20 pA/pF, n = 24) to 5.86 pA/pF in SNx myocytes (interquartile range 7.32 pA/pF, n = 21, P: < 0.005). The kinetics of inactivation of I(to) were altered after SNx with slowing both of the onset and the recovery from inactivation. The mean time constant of inactivation at +30 mV after SNx was 14.2 +/- 1.6 ms (n = 20) compared with control values of 9.8 +/- 0.6 ms (n = 23, P: < 0.05). Neither I(so) nor inward rectifier K(+) currents were altered after SNx. The action potential duration (APD(50)) at the left ventricular base was approximately 20% shorter (P: < 0.02) in hearts from SNx rats compared with controls. 4-AP (2 mM) prolonged the APD(50) in all regions in hearts from both SNx and control rats and abolished the APD(50) shortening in SNx. These results indicate that abnormalities of the cardiac transient outward K(+) current contribute to alterations in the cardiac action potential in renal failure and warrant further investigation because they may contribute to altered repolarization and arrythmogenesis.

Action Potentials↗