Weill Cornell Medicine, Department of Anesthesiology, 1300 York Avenue, New York, NY, 10065, USA.
University of Groningen, Groningen, Netherlands.
Nat Commun. 2020 Dec 16;11(1):6401. doi: 10.1038/s41467-020-20104-4.
SthK, a cyclic nucleotide-modulated ion channel from Spirochaeta thermophila, activates slowly upon cAMP increase. This is reminiscent of the slow, cAMP-induced activation reported for the hyperpolarization-activated and cyclic nucleotide-gated channel HCN2 in the family of so-called pacemaker channels. Here, we investigate slow cAMP-induced activation in purified SthK channels using stopped-flow assays, mutagenesis, enzymatic catalysis and inhibition assays revealing that the cis/trans conformation of a conserved proline in the cyclic nucleotide-binding domain determines the activation kinetics of SthK. We propose that SthK exists in two forms: trans Pro300 SthK with high ligand binding affinity and fast activation, and cis Pro300 SthK with low affinity and slow activation. Following channel activation, the cis/trans equilibrium, catalyzed by prolyl isomerases, is shifted towards trans, while steady-state channel activity is unaffected. Our results reveal prolyl isomerization as a regulatory mechanism for SthK, and potentially eukaryotic HCN channels. This mechanism could contribute to electrical rhythmicity in cells.
来自疏螺旋体（Spirochaeta thermophila）的 SthK 是一种环核苷酸调节的离子通道，在 cAMP 增加时缓慢激活。这让人想起所谓起搏通道家族中，超极化激活和环核苷酸门控通道 HCN2 的 cAMP 诱导缓慢激活。在这里，我们使用停流测定、突变、酶催化和抑制测定来研究纯化的 SthK 通道中的缓慢 cAMP 诱导激活，结果表明环核苷酸结合域中保守脯氨酸的顺/反构象决定了 SthK 的激活动力学。我们提出 SthK 存在两种形式：高配体结合亲和力和快速激活的顺式 Pro300 SthK，以及低亲和力和缓慢激活的反式 Pro300 SthK。通道激活后，脯氨酰异构酶催化的顺/反平衡向反式方向移动，而稳态通道活性不受影响。我们的结果揭示了脯氨酰异构化是 SthK 潜在的真核 HCN 通道的调节机制。这种机制可能有助于细胞中的电节律性。
