Dataset Information

Activity-dependent regulation of T-type calcium channels by submembrane calcium ions.

ABSTRACT: Voltage-gated Ca2+ channels are involved in numerous physiological functions and various mechanisms finely tune their activity, including the Ca2+ ion itself. This is well exemplified by the Ca2+-dependent inactivation of L-type Ca2+ channels, whose alteration contributes to the dramatic disease Timothy Syndrome. For T-type Ca2+ channels, a long-held view is that they are not regulated by intracellular Ca2+. Here we challenge this notion by using dedicated electrophysiological protocols on both native and expressed T-type Ca2+ channels. We demonstrate that a rise in submembrane Ca2+ induces a large decrease in T-type current amplitude due to a hyperpolarizing shift in the steady-state inactivation. Activation of most representative Ca2+-permeable ionotropic receptors similarly regulate T-type current properties. Altogether, our data clearly establish that Ca2+ entry exerts a feedback control on T-type channel activity, by modulating the channel availability, a mechanism that critically links cellular properties of T-type Ca2+ channels to their physiological roles.

SUBMITTER: Cazade M

PROVIDER: S-EPMC5308894 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Activity-dependent regulation of T-type calcium channels by submembrane calcium ions.

Cazade Magali M Bidaud Isabelle I Lory Philippe P Chemin Jean J

eLife 20170121

Voltage-gated Ca2+ channels are involved in numerous physiological functions and various mechanisms finely tune their activity, including the Ca2+ ion itself. This is well exemplified by the Ca2+-dependent inactivation of L-type Ca2+ channels, whose alteration contributes to the dramatic disease Timothy Syndrome. For T-type Ca2+ channels, a long-held view is that they are not regulated by intracellular Ca2+. Here we challenge this ...[more]

PMID: 28109159

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Project description:Neuronal excitation-transcription (E-T) coupling pathways can be initiated by local increases of Ca2+ concentrations within a nanodomain close to the L-type voltage-gated Ca2+ channel (LTCC). However, molecular mechanisms controlling LTCC organization within the plasma membrane that help creation these localized signaling domains remain poorly characterized. Here, we report that neuronal depolarization increases CaV1.3 LTCC clustering in cultured hippocampal neurons. Our previous work showed that binding of the activated catalytic domain of Ca2+/calmodulin-dependent protein kinase II (CaMKII) to an RKR motif in the N-terminal cytoplasmic domain of CaV1.3 is required for LTCC-mediated E-T coupling. We tested whether multimeric CaMKIIα holoenzymes can bind simultaneously to co-expressed CaV1.3 α1 subunits with two different epitope tags. Co-immunoprecipitation assays from HEK293T cell lysates revealed that CaMKIIα assembles multimeric CaV1.3 LTCC complexes in a Ca2+/calmodulin-dependent manner. CaMKII-dependent assembly of multi-CaV1.3 complexes is further facilitated by co-expression of the CaMKII-binding LTCC β2a subunit, relative to the β3 subunit, which cannot bind directly to CaMKII. Moreover, clustering of surface localized CaV1.3 α1 subunits in intact HEK293 cells was increased by pharmacological LTCC activation, but only in the presence of co-expressed wild-type CaMKIIα. Moreover, depolarization-induced clustering of surface-expressed CaV1.3 LTCCs in cultured hippocampal neurons was disrupted by suppressing the expression of CaMKIIα and CaMKIIβ using shRNAs. The CaMKII-binding RKR motif is conserved in the N-terminal domain of CaV1.2 α1 subunits and we found that activated CaMKIIα promoted the assembly of CaV1.2 homomeric complexes, as well as CaV1.3-CaV1.2 heteromeric complexes in vitro. Furthermore, neuronal depolarization enhanced the clustering of surface-expressed CaV1.2 LTCCs, and enhanced the colocalization of endogenous CaV1.2 LTCCs with surface-expressed CaV1.3, by CaMKII-dependent mechanisms. This work indicates that CaMKII activation-dependent LTCC clustering in the plasma membrane following neuronal depolarization may be essential for the initiation of a specific long-range signal to activate gene expression.

Project description:Key pointsTetraspanin (TSPAN) proteins regulate many biological processes, including intracellular calcium (Ca2+ ) handling. TSPAN-7 is enriched in pancreatic islet cells; however, the function of islet TSPAN-7 has not been identified. Here, we characterize how β-cell TSPAN-7 regulates Ca2+ handling and hormone secretion. We find that TSPAN-7 reduces β-cell glucose-stimulated Ca2+ entry, slows Ca2+ oscillation frequency and decreases glucose-stimulated insulin secretion. TSPAN-7 controls β-cell function through a direct interaction with L-type voltage-dependent Ca2+ channels (CaV 1.2 and CaV 1.3), which reduces channel Ca2+ conductance. TSPAN-7 slows activation of CaV 1.2 and accelerates recovery from voltage-dependent inactivation; TSPAN-7 also slows CaV 1.3 inactivation kinetics. These findings strongly implicate TSPAN-7 as a key regulator in determining the set-point of glucose-stimulated Ca2+ influx and insulin secretion.AbstractGlucose-stimulated insulin secretion (GSIS) is regulated by calcium (Ca2+ ) entry into pancreatic β-cells through voltage-dependent Ca2+ (CaV ) channels. Tetraspanin (TSPAN) transmembrane proteins control Ca2+ handling, and thus they may also modulate GSIS. TSPAN-7 is the most abundant islet TSPAN and immunostaining of mouse and human pancreatic slices shows that TSPAN-7 is highly expressed in β- and α-cells; however, the function of islet TSPAN-7 has not been determined. Here, we show that TSPAN-7 knockdown (KD) increases glucose-stimulated Ca2+ influx into mouse and human β-cells. Additionally, mouse β-cell Ca2+ oscillation frequency was accelerated by TSPAN-7 KD. Because TSPAN-7 KD also enhanced Ca2+ entry when membrane potential was clamped with depolarization, the effect of TSPAN-7 on CaV channel activity was examined. TSPAN-7 KD enhanced L-type CaV currents in mouse and human β-cells. Conversely, heterologous expression of TSPAN-7 with CaV 1.2 and CaV 1.3 L-type CaV channels decreased CaV currents and reduced Ca2+ influx through both channels. This was presumably the result of a direct interaction of TSPAN-7 and L-type CaV channels because TSPAN-7 coimmunoprecipitated with both CaV 1.2 and CaV 1.3 from primary human β-cells and from a heterologous expression system. Finally, TSPAN-7 KD in human β-cells increased basal (5.6 mM glucose) and stimulated (45 mM KCl + 14 mM glucose) insulin secretion. These findings strongly suggest that TSPAN-7 modulation of β-cell L-type CaV channels is a key determinant of β-cell glucose-stimulated Ca2+ entry and thus the set-point of GSIS.

Dataset Information

Activity-dependent regulation of T-type calcium channels by submembrane calcium ions.

Publications

Activity-dependent regulation of T-type calcium channels by submembrane calcium ions.

Similar Datasets

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