Mol Biol Rep. 2014 Jan;41(1):297-307. doi: 10.1007/s11033-013-2862-2.
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a critical role in a broad range of cell types, but the expression of the various HCN isoforms is still poorly understood. In the present study we have compared the expression of HCN isoforms in rat excitable and non-excitable tissues at both the mRNA and protein levels. Real-time PCR and Western blot analysis revealed distinct expression patterns of the four HCN isoforms in brain, heart, pituitary and kidney, with inconsistent mRNA-protein expression correlation. The HCN2 was the most abundant mRNA transcript (95.6, 78.0 and 59.0 % in kidney heart and pituitary, respectively) except in the brain (42.0 %) whereas HCN4 was the most abundant protein isoform. Our results suggest that HCN channels are mostly produced by the HCN4 isoform in heart, which contrasts with the sharp differences in the isoform stoichiometry in pituitary (15 HCN4:2 HCN2:1 HCN1:1 HCN3), kidney (24 HCN4:2 HCN3:1 HCN2:1 HCN1) and brain (3 HCN4:2 HCN2:1 HCN1:1 HCN3). Moreover, deviations of the electrophoretic molecular weight (MW) of the HCN isoforms relative to the theoretical MW were observed, suggesting that N-glycosylation and enzymatic proteolysis influences HCN channel surface expression. We hypothesize that selective cleavage of HCN channels by membrane bound metalloendopeptidases could account for the multiplicity of properties of native HCN channels in different tissues.
超极化激活环核苷酸门控 (HCN) 通道在广泛的细胞类型中发挥着关键作用，但各种 HCN 同工型的表达仍知之甚少。在本研究中，我们比较了大鼠兴奋和非兴奋组织中 HCN 同工型在 mRNA 和蛋白质水平上的表达。实时 PCR 和 Western blot 分析显示，在脑、心、垂体和肾中，四种 HCN 同工型的表达模式存在明显差异，mRNA-蛋白表达相关性不一致。HCN2 是最丰富的 mRNA 转录本（肾、心、垂体中分别为 95.6%、78.0%和 59.0%，除脑中外为 42.0%），而 HCN4 是最丰富的蛋白同工型。我们的结果表明，HCN 通道主要由心脏中的 HCN4 同工型产生，这与垂体中同工型比例的明显差异形成对比（15 HCN4:2 HCN2:1 HCN1:1 HCN3）、肾（24 HCN4:2 HCN3:1 HCN2:1 HCN1）和脑（3 HCN4:2 HCN2:1 HCN1:1 HCN3）。此外，观察到 HCN 同工型的电泳分子量（MW）相对于理论 MW 的偏差，表明 N-糖基化和酶蛋白水解影响 HCN 通道表面表达。我们假设，膜结合金属内肽酶对 HCN 通道的选择性切割可以解释不同组织中天然 HCN 通道多种特性的原因。
