Department of Chemical Physiology and Biochemistry, Oregon Health & Science University, Portland, Oregon, USA.
Krannert Institute of Cardiology, Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA.
Physiol Rep. 2021 Nov;9(22):e15121. doi: 10.14814/phy2.15121.
Variants in the LMNA gene, which encodes for Lamin A/C, are associated with cardiac conduction disease (CCD). We previously reported that Lamin A/C variants p.R545H and p.A287Lfs*193, which were identified in CCD patients, decreased peak I in HEK-293 cells expressing Na 1.5. Decreased peak I in the cardiac conduction system could account for patients' atrioventricular block. We found that serine 22 (Ser 22) phosphorylation of Lamin A/C was decreased in the p.R545H variant and hypothesized that lamin phosphorylation modulated Na 1.5 activity. To test this hypothesis, we assessed Na 1.5 function in HEK-293 cells co-transfected with LMNA variants or treated with the small molecule LBL1 (lamin-binding ligand 1). LBL1 decreased Ser 22 phosphorylation by 65% but did not affect Na 1.5 function. To test the complete loss of phosphorylation, we generated a version of LMNA with serine 22 converted to alanine 22 (S22A-LMNA); and a version of mutant R545H-LMNA that mimics phosphorylation via serine 22 to aspartic acid 22 substitution (S22D-R545H-LMNA). We found that S22A-LMNA inhibited Lamin-mediated activation of peak I by 63% and shifted voltage-dependency of steady-state inactivation of Na 1.5. Conversely, S22D-R545H-LMNA abolished the effects of mutant R545H-LMNA on voltage-dependency but not peak I . We conclude that Lamin A/C Ser 22 phosphorylation can modulate Na 1.5 function and contributes to the mechanism by which R545H-LMNA alters Na 1.5 function. The differential impact of complete versus partial loss of Ser 22 phosphorylation suggests a threshold of phosphorylation that is required for full Na 1.5 modulation. This is the first study to link Lamin A/C phosphorylation to Na 1.5 function.
LMNA 基因编码核纤层蛋白 A/C，其变体与心脏传导疾病（CCD）相关。我们之前报道过，在 CCD 患者中发现的核纤层蛋白 A/C 变体 p.R545H 和 p.A287Lfs*193 会降低表达 Na 1.5 的 HEK-293 细胞中的峰值 I。心脏传导系统中峰值 I 的降低可能导致患者发生房室传导阻滞。我们发现，p.R545H 变体中核纤层蛋白 A/C 的丝氨酸 22（Ser 22）磷酸化减少，并且假设核纤层蛋白磷酸化调节 Na 1.5 活性。为了验证该假设，我们评估了 HEK-293 细胞中 LMNA 变体共转染或用小分子 LBL1（核纤层结合配体 1）处理后的 Na 1.5 功能。LBL1 将 Ser 22 磷酸化降低了 65%，但不影响 Na 1.5 功能。为了测试完全失去磷酸化，我们生成了一种 Ser 22 突变为丙氨酸 22（S22A-LMNA）的 LMNA 变体；以及一种通过丝氨酸 22 突变为天冬氨酸 22 模拟磷酸化的 R545H-LMNA 突变体（S22D-R545H-LMNA）。我们发现，S22A-LMNA 抑制 Lamin 介导的峰值 I 激活达 63%，并改变 Na 1.5 的稳态失活的电压依赖性。相反，S22D-R545H-LMNA 消除了突变体 R545H-LMNA 对电压依赖性的影响，但对峰值 I 没有影响。我们得出结论，核纤层蛋白 A/C 的 Ser 22 磷酸化可以调节 Na 1.5 的功能，并有助于解释 R545H-LMNA 改变 Na 1.5 功能的机制。完全或部分丧失 Ser 22 磷酸化的差异影响表明需要达到完全 Na 1.5 调节的磷酸化阈值。这是首次将核纤层蛋白 A/C 磷酸化与 Na 1.5 功能联系起来的研究。
