ROMK1鉀離子通道受磷酸化與抗癲癇藥物調控分子機轉之探討

Molecular mechanism of ROMK1 channels regulation by phosphorylation and anticonvulsants

李建興(Chien-Hsing Lee)

指導教授 ： 劉宏輝

國立臺灣大學/醫學院/藥理學研究所/博士(2008年)

https://doi.org/10.6342/NTU.2008.01033

摘要

ROMK1鉀離子通道廣泛分佈於體內，調控許多重要的生理功能，包括：維持細胞靜止膜之電位，神經突觸之興奮性，和腎臟鉀離子的運輸。已知PKA的磷酸化過程可以來調控ROMK1鉀離子通道，近年來有研究指出，PKA調控ROMK1鉀離子通道是藉由於PIP2的參與。ROMK1鉀離子通道也被證明會受到細胞內酸鹼值影響，在細胞內酸性pHi值下，引起ROMK1鉀離子通道的關閉，測得ROMK1鉀離子通道的有效酸性解離常數為6.85。在本實驗中，我們利用inside-out電位鉗定的電生理方式，記錄表現在蛙卵上(Xenopous oocytes)的ROMK1鉀離子通道。PKA磷酸化降低離子通道對細胞內酸鹼值的靈敏度，有效酸性解離常數往較酸性數值偏移(~6.58)。由分子動力學模擬結果表示，當ROMK1鉀離子通道受到PKA磷酸化作用，會產生一個穩定的過渡狀態，縮短PKA磷酸化作用位點S219和PIP2結合位點R217之間的距離，使PIP2結合位點K218向細胞膜移動，這樣結構之變化，導致在離子通道上更容易向細胞膜上的PIP2結合。只有當ROMK1鉀離子通道受到PKA磷酸化作用後，PIP2得以劑量依賴性方式重新活化因酸化而引起活性受抑制的通道。這結果意味著PKA次序性地透過通道結構之變化，以利增加通道與PIP2的相互作用，而調控ROMK1鉀離子通道對細胞內酸鹼值的靈敏度。因此，我們提出了PKA磷酸化作用調控離子通道對細胞內酸鹼值的敏感性之分子機制。
已知ROMK1鉀離子通道的活性受到PKC磷酸化作用而抑制。實驗結果發現由PMA活化PKC磷酸化作用，不僅影響通道對細胞內酸鹼值的靈敏度，並且調控PIP2與離子通道的相互作用。ROMK1鉀離子通道具有六個假定的PKC磷酸化作用位點(S4，S183，T191，T193，S201和T234)。而S183已報導不是PKC磷酸化的位點。我們實驗結果發現，PKC磷酸化作用位點突變的離子通道，S4，T191和S201不影響離子通道對細胞內酸鹼值的敏感性及與PIP2的相互作用。T193和T234不僅調控離子通道對細胞內酸鹼值的敏感性，且影響通道與PIP2的相互作用。我們的結論，PKC磷酸化作用位點T193和T234參與調控離子通道對細胞內酸鹼值的靈敏度，且可透過影響離子通道與PIP2相互作用途徑而導致。
透過建立ROMK1 鉀離子通道同源結構，結合單點突變設計和電生理分析方式。我們的結果顯示，一群帶正電荷的氨基酸(R188，R217，和K 218)在一個適當的距離(~13 Å)而形成一個平面，與細胞膜上的PIP2相互作用。在作用平面區域上下的帶正電荷氨基酸(K187和R212A)，無法與PIP2相互作用和改變對細胞內酸鹼值的靈敏度。進一步發現，在作用平面區域上的不帶電荷氨基酸(I192，N215，L216，S219 和L220)，亦影響ROMK1鉀離子通道與PIP2的相互作用及調控離子通道對細胞內酸鹼值的靈敏度。在內流型整流鉀離子通道上，我們發現高度保有這一群帶正電荷的氨基酸所聚集形成之作用平面區域。
除了探討ROMK1鉀離子通道受磷酸化控制之分子生理機制外，我們也進一步研究影響ROMK1鉀離子通道活性之藥物機轉。ROMK1鉀離子通道密集分布於海馬迴，具有穩定膜電位的功能。鉀離子通道活性已證實受到許多在臨床上廣泛使用的抗癲癇用藥調控。然而，新一代抗癲癇藥物gabapentin，pregabalin及levetiracetam是否與ROMK1鉀離子通道作用機轉尚未瞭解。我們研究證明這些新一代抗癲癇藥物以dose-dependent 方式活化ROMK1 鉀離子通道。活化機轉是與細胞內pH值及PIP2調節無關，而受到PKA磷酸化之調控。

關鍵字

ROMK1鉀離子通道 ； 蛋白激酶細胞膜脂質 ； 細胞內酸鹼值 ； 抗癲癇用藥

並列摘要

ROMK1 (Kir1.1) channels are widely distributed in different tissues and regulate many important cellular processes, including the membrane resting potentials, cell and synaptic excitability, and the renal K+ transport. ROMK1 channels are mediated by intracellular pH (pHi), phosphorylation by kinases (PKA and PKC) and phosphatidylinositol 4, 5-bisphosphate (PIP2).
Part 1: The regulatory mechanism of ROMK1 channels. We investigate the role of protein kinase A (PKA) in the pHi gating of ROMK1 channels. Using giant patch clamp with Xenopus oocytes expresses wild-type and mutant ROMK1 channels. We investigated between the role of PKA in regulation of ROMK1 channels by pHi and the mechanisms. PKA-mediated phosphorylation decreased the sensitivity of ROMK1 channels to pHi. Molecular dynamics simulations suggest a stable transition state in which the shortening of distance between S219 and R217 and the movement of K218 towards the membrane after the PKA-phosphorylation, which conformational changes resulted in the PIP2 binding residues more accessible to the membrane-bound PIP2. PIP2 dose-dependently reactivates the acidification-induced rundown channels only when ROMK1 channels have been phosphorylated by PKA. It implies a sequence regulatory mechanism in the pHi gating of ROMK1 channels in which the channel-PIP2 interaction may be enhanced through PKA-mediated phosphorylation. We therefore propose a molecular mechanism of the PKA-mediated phosphorylation in regulating the pHi gating of ROMK1 channels.
We investigated the role of PKC in regulation of ROMK1 channels by pHi and the mechanisms. Activation of PKC by phorbol myristate acetate (PMA) on ROMK1 channels not only increases its sensitivity to pHi but also reduces PIP2-channels interactions. ROMK1 channels have been five putative PKC phosphorylation sites, S4 and S201, as well as T191, T193 and T234. T193 and T234 are the two main PKC phosphorylation sites that are important for the effect of pHi sensitivity of ROMK1 channels. T193A decreases pHi sensitivity and increases PIP2 interaction with the channels, whereas T193D that mimic the negative charge carried by a phosphate group bound to a serine increases pHi sensitivity. T234A, however, increases pHi sensitivity and reduces PIP2-channels interaction. We conclude that PKC regulates the ROMK1 activity channel to intracellular protons by affecting channel-PIP2 interaction.
We perform the experiments in ROMK1 pHi-gating with electrophysiology combined with mutational and structural analysis. The mutants design is based upon previous discoveries regarding the interaction with PIP2 and our present structure model of ROMK1. Our results suggest the importance function of a cluster of basic residues (R188, R217and K218) that form a plane in an appropriate distance (~13 Å) to interact with membrane PIP2. Basic residues (K187 and R212) above and below this plane discriminates on the regulation of this interaction and pHi-gating. Several non basic residues (I192, N215, L216, S219 and L220) in this plane have been found to mediate channel-PIP2 interaction and pHi gating. This plane form by a cluster of basic residues can be observed with high conservation in the Kir channel family.
Part 2: we investigated ROMK1 chhannels are modulated by antiepileptic drugs (AEDs) and the mechanisms. Gabapentin (GBP), pregabalin (PGB) and levetiracetam (LEV) are an effective anticonvulsant in treating seizures. We explored the mechanisms underling those AEDs on ROMK1 channels expressed in Xenopus oocytes by an inside-out patch-clamp recording. Those AEDs increased ROMK1 channels activity in a concentration-dependent manner. Those AEDs increases both wide-type and pHi critical residue mutant (K80M) channels activity in different pHi values, indicating that the activatory effect is independent from pHi. Those AEDs does not influence the enhancement effect of channels activity on the wild type is similar to the PIP2-binding sites mutated channels (R188Q, R217A and K218A), suggesting those AEDs may not activate ROMK1 channels through PIP2 pathway. Those AEDs fails to enhance the channel activity in the presence of PKA inhibitor (H89 or KT 5720) indicating that PKA-mediate phosphorylation participates in the function of GBP. This observation is further supported by the evidence that Those AEDs has no effect on the PKA-phosphorylation sites mutated channels (S219A and S313A). Moreover, Those AEDs could not activate the constructed mutants (S219D and S313D) that mimic the negative charge carried by a phosphate group bound to a serine, imply that the effect of PKA in those AEDs may induce conformational modification but not charge-charge interaction in ROMK1 channels. The activity of PKA was increased in epilepsy. ROMK1 channels may be phosphorylated by PKA during seizure which provided an opportunity for those AEDs enhancing ROMK1 channels activity and restore neuronal RMP to prevent seizure spreading.

並列關鍵字

ROMK1 channels ； PKA ； PKC ； PIP2 ； pHi ； antiepileptic drugs

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