壓力荷爾蒙對鉀離子通道之調控

Abstract

當一個基因被轉錄成mRNA 的過程中必需經過剪接，而百分之七十五的基因其 mRNA 均有多種不同的剪接方式，所以同一條基因轉譯出來的蛋白質會在功能上有一些差異。這種一基因多用途的設計，對於生物體來講，是非常經濟的作法。雖然這種 mRNA 的選擇性剪接之例子很多，但是對於其調控機制的了解卻很少。我之前已證明壓力荷爾蒙可以調控slo 基因的 mRNA選擇性剪接。這個研究計畫是我先前研究的延伸，主要目的為探討壓力荷爾蒙如何調控基因轉錄成mRNA之選擇性剪接。 slo 基因轉譯之蛋白質為一鉀離子通道，由腎上腺皮質所分泌之壓力荷爾蒙可調控其mRNA選擇性剪接。在slo基因的第五個選擇性剪接的位子可以有二種剪接方式: 一種剪接完包含額外的174 個核苷酸稱為 STREX，另一種接完不含任何額外的核苷酸稱為ZERO。細胞如果有STREX 型的離子通道，將會較能連續地產生膜電位改變，如果是釋放腎上腺素的細胞將能在緊急狀況下快速釋放大量腎上腺素，所以細胞對外來刺激的反應快慢及強度是可以藉由調整slo基因的mRNA選擇性剪接來改變。所以之前的壓力是會透過壓力荷爾蒙來調整slo基因的mRNA選擇性剪接而影響將來細胞對壓力的反應。為了探討鈣離子是否為壓力荷爾蒙調控選擇性剪接之傳訊物，於是我設計了以下實驗: (1) 氯化鉀溶液所引起的膜電位去極化造成鈣離子增加是否也可改變嗜鉻細胞之slo基因的mRNA選擇性剪接？(2)是否壓力荷爾蒙也可以造成細胞內鈣離子濃度改變? (3)改變細胞內鈣離子濃度可否改變slo基因的mRNA選擇性剪接? (4)利用鈣離子通道阻斷藥物來避免細胞內鈣離子濃度改變，是否可以阻斷氯化鉀溶液或壓力荷爾蒙所引發的slo基因的mRNA選擇性剪接之改變? (5)測試鉀離子結合蛋白是否也在slo基因的mRNA選擇性剪接過程扮演重要角色。

Alternative splicing allows a single gene to give rise to multiple mRNA variants each encoding proteins with different properties. It is estimated that transcripts of more than 75% of human genes are alternative spliced. I have previously demonstrated that stress hormones differentially regulate the alternative splicing of slo gene transcripts. The main objective of this proposal is to identify downstream messengers involved in the regulation of stress hormone induced alternative splicing. Slo gene encoded voltage- and Ca-activated BK channels generate their functional diversity through both protein modification and alternative splicing. In both adrenal and pituitary glands, stress hormones (adrenocorticoids) regulate alternative splicing of the slo transcript. Two splice variants differing in splice site 5 of the slo transcript have been identified. “STREX” contains a 174 nucleotide exon at this site, while “ZERO” lacks the exon. STREX type channels are activated at more negative voltage than the ZERO type. Therefore STREX channels have faster sodium channel recovery time after activation. These properties increase a cell’s capability for repetitive firing and could facilitate rapid secretion of epinephrine by adrenal chromaffin cells during a “crisis” situation. Thus, experiences of stress, by stimulating the release of stress hormones which lead to changes in the alternative splicing of transcripts encoding BK channels, might have a lasting effect on future chromaffin cell excitability and adrenaline release. I have also shown that the androgen group of adrenocorticoids, including DHEA and testosterone increases STREX exon inclusion, while cortisol and aldosterone decreases STREX exon inclusion in bovine adrenal chromaffin cells. Thus, alternative splicing of slo is regulated by a combination of stress hormonal inputs. This multifactorial regulation provides flexibility in modifying the intrinsic excitability of cells, and help prepares the cells for future response to stress. In cultured pituitary cells, KCl-mediated depolarization decreases STREX exon inclusion. Since, KCl-mediated depolarization raises intracellular calcium levels, calcium is a likely second messenger in pituitary alternative splicing. These observations suggest: 1) Ca may be an important regulatory factor in slo splicing in chromaffin cells, and 2)Ca may play a key intermediary role in the effects of steroids on slo splicing. This proposal addresses several unanswered questions regarding the mechanism of hormone-induced alternative splicing: (1) Does depolarization lead to changes in the alternative splicing pattern of slo transcripts in adrenal chromaffin cells. If so, is calcium the second messenger in mediating the changes. (2) Can intracellular calcium levels be changed by stress hormones and KCl-mediated depolarization in adrenal chromaffin cells? (3) Is an increase in intracellular calcium level sufficient for enhancing inclusion of the STREX exon? (4) Are increases in intracellular calcium level necessary for KCl and stress hormones induced alternative splicing. (5) What are the calcium-binding proteins that may be involved in the regulation of STREX exon inclusion. If calcium proves to be a critical regulator of slo splicing in chromaffin cells, this is evidence that in addition to the multifactoral regulation by hormones, electrical activity triggered by splanchnic nerve inputs to chromaffin cells provides another important pathway for experience-dependent plasticity in the excitable properties of chromaffin cells.