| Abstract: | 電壓敏感型鈣離子通道由α1、α2δ、β與γ次單元組成，α1是構成孔道的次單元，α2δ、β與γ則是輔助用的次單元。α2δ與β次單元功能上為正向調控因子，會幫助α1運送到細胞膜上並加強通道的活化。相反的，實驗證據顯示γ次單元功能上分歧不一。在十個γ次單元中，γ1與γ6屬於通道的負向調控因子，抑制肌肉細胞的鈣離子通道電流。另一方面，γ2、γ3、γ4、γ5、γ7 及γ8 被認為是transmembrane AMPA receptor regulatory proteins (TARPs)。至於TMEM114與TMEM235(近來被發現的γ家族成員)，其生理功能仍了解甚少。為何這十個γ基因是如此相似但他們生理功能卻有相當大的分歧?由於現有的實驗證據無法提供充足的線索，因此我們轉向利用生物資訊方法進行分析，試圖推敲出γ基因家族的演化歷程。藉由在26個物種進行protein-protein BLAST，發現這群calcium channel γ (CACNG)基因在八目鰻與硬骨魚之間，數量倍增而演化出現存的十個CACNG分子，也各自獨立分成4個單系群。在脊椎動物演化過程，γ基因在不同的物種中獨立地消失與複製。雖然我們親緣關係樹分析基本上與先前結果一致，但是無脊椎動物序列之存在顯示脊椎與無脊椎動物的γ基因擁有共同祖先，最早可以追溯到兩側對稱動物。有趣的是，在染色體地圖(chromosome map)中，PKC 跟γ基因緊鄰在一起，意味著這群蛋白可能跟鈣離子的濃度恆定或蛋白質磷酸化有關。TMEM114及TMEM235也緊鄰GRIN基因及CACNA基因，意指TMEM114及TMEM235可能與GRIN及CACNA有功能上的交互作用。藉由搜尋在CACNG基因附近的同源染色體片段，我們修正了先前提出的CACNG基因演化途徑。在演化速率分析中，(γ4,γ8)及(TMEM114,TMEM235)在第二次染色體複製後，展現出顯著的氨基酸非同義置換，暗示他們可能在動物中演化出其分岐功能。此外，在硬骨魚中多出一套的γ(γ1,γ2,γ3,γ5及γ7)，顯示他們也可能獲得新的功能。在此一研究中，我們希望可以洞悉γ基因的演化歷史，解釋現今動物γ功能的差異性，進而提供我們未來以實驗方法驗證γ基因功能時實驗設計上的洞見。
Voltage-dependent calcium channels (VDCCs) are comprised of pore-forming α1 subunits as well as three other auxiliary subunits: α2δ, β and γ subunits. The α2δ and β subunits are positive regulators of VDCCs that enhance membrane insertion of the α1 subunits and channel activation. In contrast, the functions of the γ subunits are not completely established, because experimental data have suggested functional diversity. Out of the ten members of the γ subunits, γ1 and γ6 are negative regulators of VDCCs that inhibit calcium current in muscle cells. In contrast, the γ2, γ3, γ4, γ5, γ7 and γ8 are known as the transmembrane AMPA receptor regulatory proteins (TARPs). As for TMEM114 and TMEM235 (two new members of the family), their physiological functions remain largely unknown. While these ten γ genes are the closest homologs within mammalian genomes, why are their functions so diverse? Because experimental paradigms have not provided enough clues, we turn to bioinformatical analysis for evolutionary insight. By conducting protein-protein BLAST between twenty-six animal species, we found that several γ genes emerged by gene and chromosome duplications between hyperoartia and osteichthyes, evolving into the ten currently known γ genes that are clustered into four monophyletic groups in vertebrate. In vertebrate lineages, γ genes were independently lost and duplicated. Although our phylogenetic analysis is consistent with previous results, the invertebrate sequences demonstrate that vertebrate and invertebrate γ’s share common ancestors in as far back as the bilaterians. Interestingly, γ genes are almost always associated with PKC genes on chromosome, suggesting that the functions of γ proteins are related to the homeostasis of calcium or protein phosphorylation. TMEM114 and TMEM235 genes are closely located with GRIN and CACNA1 genes on chromosome, implying that TMEM114 and TMEM235 may functionally interact with GRIN and CACNA1. By searching the paralogous chromosome segments around CACNG genes, we revised the evolution pathways that was previously proposed. In evolutionary rate analysis, (γ4, γ8) and (TMEM114, TMEM235) exhibited significantly nonsynonymous substitution after the 2nd round of chromosome duplication, implying that their functions have diverged in the animal lineage. In addition, the additional copies of γ (γ1, γ2, γ3, γ5 and γ7) in osteichthyes may have acquired novel functions. By elucidating the historical events that produced these ten γ genes, we hope to contribute to the explanation of the functional diversity of calcium channel γ subunits and to provide insight for the experimental design of functional verification of the ten γ proteins in the future. |
