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Glucose-6-phosphate dehydrogenase increases Ca²⁺ currents by interacting with Ca_v1.2 and reducing intrinsic inactivation of the L-type calcium channel.

ABSTRACT: Pyridine nucleotides, such as NADPH and NADH, are emerging as critical players in the regulation of heart and vascular function. Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, is the primary source and regulator of cellular NADPH. In the current study, we have identified two isoforms of G6PD (slow and fast migrating) and functionally characterized the slow migrating isoform of G6PD (G6PD₅₄₅) in bovine and human arteries. We found that G6PD₅₄₅ is eluted in the caveolae fraction of vascular smooth muscle (VSM) and has a higher maximum rate of reaction (V_max: 1.65-fold) than its fast migrating isoform (G6PD₅₁₅). Interestingly, caveolae G6PD forms a complex with the pore-forming α_1C-subunit of the L-type Ca²⁺ channel, Ca_v1.2, as demonstrated by a proximity ligation assay in fixed VSMCs. Additionally, Förster resonance energy transfer (FRET) analysis of HEK293-17T cells cotransfected with red fluorescent protein (RFP)-tagged G6PD₅₄₅ (C-G6PD₅₄₅) and green fluorescent protein (GFP)-tagged Ca_v1.2-(Ca_v1.2-GFP) demonstrated strong FRET signals as compared with cells cotransfected with Ca_v1.2-GFP and C-G6PD₅₁₅. Furthermore, L-type Ca²⁺ channel conductance was larger and the voltage-independent component of availability (c₁) was augmented in C-G6PD₅₄₅ and Ca_v1.2-GFP cotransfectants compared with those expressing Ca_v1.2-GFP alone. Surprisingly, epiandrosterone, a G6PD inhibitor, disrupted the G6PD-Ca_v1.2 complex, also decreasing the amplitude of L-type Ca²⁺ currents and window currents, thereby reducing the availability of the c₁ component. Moreover, overexpression of adeno-G6PD₅₄₅-GFP augmented the KCl-induced contraction in coronary arteries compared with control. To determine whether overexpression of G6PD had any clinical implication, we investigated its activity in arteries from patients and rats with metabolic syndrome and found that G6PD activity was high in this disease condition. Interestingly, epiandrosterone treatment reduced elevated mean arterial blood pressure and peripheral vascular resistance in metabolic syndrome rats, suggesting that the increased activity of G6PD augmented vascular contraction and blood pressure in the metabolic syndrome. These data suggest that the novel G6PD-Ca_v1.2 interaction, in the caveolae fraction, reduces intrinsic voltage-dependent inactivation of the channel and contributes to regulate VSM L-type Ca²⁺ channel function and Ca²⁺ signaling, thereby playing a significant role in modulating vascular function in physiological/pathophysiological conditions.NEW & NOTEWORTHY In this study we have identified a novel isozyme of glucose-6-phosphate dehydrogenase (G6PD), a metabolic enzyme, that interacts with and contributes to regulate smooth muscle cell l-type Ca²⁺ ion channel function, which plays a crucial role in vascular function in physiology and pathophysiology. Furthermore, we demonstrate that expression and activity of this novel G6PD isoform are increased in arteries of individuals with metabolic syndrome and in inhibition of G6PD activity in rats of metabolic syndrome reduced blood pressure.

SUBMITTER: Gupte R

PROVIDER: S-EPMC7474437 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Mammalian circadian (24 h) rhythms are timed by the pattern of spontaneous action potential firing in the suprachiasmatic nucleus (SCN). This oscillation in firing is produced through circadian regulation of several membrane currents, including large-conductance Ca2+- and voltage-activated K+ (BK) and L-type Ca2+ channel (LTCC) currents. During the day steady-state BK currents depend mostly on LTCCs for activation, whereas at night they depend predominantly on ryanodine receptors (RyRs). However, the contribution of these Ca2+ channels to BK channel activation during action potential firing has not been thoroughly investigated. In this study, we used a pharmacological approach to determine that both LTCCs and RyRs contribute to the baseline membrane potential of SCN action potential waveforms, as well as action potential-evoked BK current, during the day and night, respectively. Since the baseline membrane potential is a major determinant of circadian firing rate, we focused on the LTCCs contributing to low voltage activation of BK channels during the subthreshold phase. For these experiments, two LTCC subtypes found in SCN (CaV1.2 and CaV1.3) were coexpressed with BK channels in heterologous cells, where their differential contributions could be separately measured. CaV1.3 channels produced currents that were shifted to more hyperpolarized potentials compared with CaV1.2, resulting in increased subthreshold Ca2+ and BK currents during an action potential command. These results show that although multiple Ca2+ sources in SCN can contribute to the activation of BK current during an action potential, specific BK-CaV1.3 partnerships may optimize the subthreshold BK current activation that is critical for firing rate regulation.NEW & NOTEWORTHY BK K+ channels are important regulators of firing. Although Ca2+ channels are required for their activation in excitable cells, it is not well understood how BK channels activate using these Ca2+ sources during an action potential. This study demonstrates the differences in BK current activated by CaV1.2 and CaV1.3 channels in clock neurons and heterologous cells. The results define how specific ion channel partnerships can be engaged during distinct phases of the action potential.

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Glucose-6-phosphate dehydrogenase increases Ca²⁺ currents by interacting with Ca_v1.2 and reducing intrinsic inactivation of the L-type calcium channel.

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Glucose-6-phosphate dehydrogenase increases Ca2+ currents by interacting with Cav1.2 and reducing intrinsic inactivation of the L-type calcium channel.

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OmicsDI is part of the ELIXIR infrastructure

Tweets

Glucose-6-phosphate dehydrogenase increases Ca²⁺ currents by interacting with Ca_v1.2 and reducing intrinsic inactivation of the L-type calcium channel.