Haase Hannelore, Alvarez Julio, Petzhold Daria, Doller Anke, Behlke Joachim, Erdmann Jeanette, Hetzer Roland, Regitz-Zagrosek Vera, Vassort Guy, Morano Ingo
Max Delbrück Center for Molecular Medicine (MDC), D-13092 Berlin, Germany.
FASEB J. 2005 Dec;19(14):1969-77. doi: 10.1096/fj.05-3997com.
Defective L-type Ca2+ channel (I(CaL)) regulation is one major cause for contractile dysfunction in the heart. The I(CaL) is enhanced by sympathetic nervous stimulation: via the activation of beta-adrenergic receptors, PKA phosphorylates the alpha1C(Ca(V)1.2)- and beta2-channel subunits and ahnak, an associated 5643-amino acid (aa) protein. In this study, we examined the role of a naturally occurring, genetic variant Ile5236Thr-ahnak on I(CaL). Binding experiments with ahnak fragments (wild-type, Ile5236Thr mutated) and patch clamp recordings revealed that Ile5236Thr-ahnak critically affected both beta2 subunit interaction and I(CaL) regulation. Binding affinity between ahnak-C1 (aa 4646-5288) and beta2 subunit decreased by approximately 50% after PKA phosphorylation or in the presence of Ile5236Thr-ahnak peptide. On native cardiomyocytes, intracellular application of this mutated ahnak peptide mimicked the PKA-effects on I(CaL) increasing the amplitude by approximately 60% and slowing its inactivation together with a leftward shift of its voltage dependency. Both mutated Ile5236Thr-peptide and Ile5236Thr-fragment (aa 5215-5288) prevented specifically the further up-regulation of I(CaL) by isoprenaline. Hence, we suggest the ahnak-C1 domain serves as physiological brake on I(CaL). Relief from this inhibition is proposed as common pathway used by sympathetic signaling and Ile5236Thr-ahnak fragments to increase I(CaL). This genetic ahnak variant might cause individual differences in I(CaL) regulation upon physiological challenges or therapeutic interventions.
L型钙通道（I(CaL)）调节缺陷是心脏收缩功能障碍的一个主要原因。交感神经刺激可增强I(CaL)：通过β-肾上腺素能受体的激活，蛋白激酶A（PKA）使α1C（Ca(V)1.2）和β2通道亚基以及ahnak（一种相关的5643个氨基酸（aa）的蛋白质）磷酸化。在本研究中，我们研究了天然存在的基因变体Ile5236Thr-ahnak对I(CaL)的作用。使用ahnak片段（野生型、Ile5236Thr突变型）的结合实验和膜片钳记录显示，Ile5236Thr-ahnak严重影响β2亚基相互作用和I(CaL)调节。PKA磷酸化后或存在Ile5236Thr-ahnak肽时，ahnak-C1（氨基酸4646 - 5288）与β2亚基之间的结合亲和力降低约50%。在天然心肌细胞上，细胞内应用这种突变的ahnak肽可模拟PKA对I(CaL)的作用，使幅度增加约60%，减缓其失活，并使其电压依赖性向左移位。突变的Ile5236Thr肽和Ile5236Thr片段（氨基酸5215 - 5288）均特异性地阻止了异丙肾上腺素对I(CaL)的进一步上调。因此，我们认为ahnak-C1结构域作为I(CaL)的生理制动器。解除这种抑制被认为是交感神经信号传导和Ile5236Thr-ahnak片段增加I(CaL)所使用的共同途径。这种基因ahnak变体可能在生理挑战或治疗干预时导致I(CaL)调节的个体差异。
