Sarcoplasmic Reticulum SR K Channel Function

描述（由申请人提供）：肌浆网（SR）Ca释放代表了一种大的电荷易位，可以非常快速（<1 ms）地将SR膜电位（Vm）移动到Ca平衡电位（ECa），在此净Ca释放将停止（1）。然而，这不会发生，因为在释放期间存在同时逆流。在上一个资助期间，我们建立了多离子，选择性差，孔的ryanodine受体（RyR）介导大的反Mg和K通量，而它释放Ca。因此RyR本身携带所需逆流的大部分。这使得SR K和Cl通道的生理作用不清楚。SR K和Cl通道在释放过程中可能携带一个小的重要逆流元素，但它们也可能为反离子在释放后重新平衡SR提供重要途径。事实上，SR离子和电荷平衡的机制知之甚少，以至于我们不知道它们对SR Ca处理的意义，它们可能的病理贡献或它们是否可以用于治疗益处。 2010年，三聚体细胞内阳离子（TRIC）蛋白被鉴定为SR K通道（2）。两种TRIC同种型（A和B）的消融是胚胎致死的。仅TRIC-A（骨骼肌中的主要形式）的消融在SR Ca处理中产生明显的异常（异常局部Ca释放事件和SR Ca过载;（3-5））。TRIC-A KO肌肉不仅证明了SR Ca处理和SR离子平衡之间存在机械联系，而且提供了一个独特的机会来定义它。在这里，我们将使用TRIC-A KO肌肉来定义通过RyR，SR K和SR Cl通道的复杂时空Ca，Mg，K和Cl通量如何控制SR离子（电压）平衡并调节骨骼肌中的SR Ca处理。 这里检验的假设是：SR K（TRIC）和SR Cl通道在个体骨骼肌SR Ca释放事件期间不携带必需的逆流，而是提供关键的SR离子再平衡途径，确保SR离子平衡并且SR Vm在释放事件之间返回到静息值。有缺陷的再平衡，而不是缺失逆流，解释了在TRIC-KO骨骼肌中观察到的SR Ca处理缺陷。这是通过以下具体目标来检验的。目的1：确定SR K或Cl通道在SR Ca释放过程中是否携带必需的逆流。目的2：确定SR K或Cl通道是否提供重要的静息SR离子再平衡途径。目的3：建立TRIC-A KO肌肉异常SR Ca处理的机制。预期结果：通过将SR功能的现有“以钙为中心”观点转变为涵盖多离子（Ca、Mg、K、Cl）现实（其控制SR Ca释放/摄取），描绘SR病理性衰竭的新潜在点（和/或SR功能可在治疗上操纵的部位）。

DESCRIPTION (provided by applicant): Sarcoplasmic reticulum (SR) Ca release represents a large charge translocation that could very quickly (<1 ms) move the SR membrane potential (Vm) to the Ca equilibrium potential (ECa), where net Ca release will cease (1). However, this does not happen because there is a simultaneous countercurrent during release. In the previous funded period, we established that the multi-ion, poorly selective, pore of the ryanodine receptor (RyR) mediates large counter Mg and K fluxes, while it releases Ca. The RyR therefore carries the bulk of required countercurrent itself. This leaves the physiological roles of the SR K and Cl channels unclear. The SR K and Cl channels may carry a small important element of countercurrent during release, but they may also provide vital pathways for counter ions to re-equilibrate across the SR after release. Indeed, the mechanisms of SR ion and charge balance are so poorly understood that we do not know their significance to SR Ca handling, their possible pathological contributions or whether they can be exploited for therapeutic benefit. In 2010, the trimeric intracellular cation (TRIC) protein was identified as the SR K channel (2). Ablation of both TRIC isoforms (A & B) is embryonically lethal. Ablation of just TRIC-A (predominant form in skeletal muscle) generates clear in SR Ca handling abnormalities (abnormal local Ca release events and SR Ca overload; (3-5)). TRIC-A KO muscle not only proves there is a mechanistic link between SR Ca handling and SR ion balance, but provides a unique opportunity to define it. Here, we will use TRIC-A KO muscle to define how the complex spatiotemporal Ca, Mg, K and Cl fluxes through RyR, SR K and SR Cl channels control SR ion (voltage) balance and modulate SR Ca handling in skeletal muscle. The hypothesis tested here is: The SR K (TRIC) and SR Cl channels do not carry essential countercurrent during individual skeletal muscle SR Ca release events but instead provides crucial SR ion re-equilibration pathways, assuring SR ion balance and that SR Vm returns to resting values between release events. Defective re-equilibration, not missing countercurrent, explains the SR Ca handling defects observed in TRIC-KO skeletal muscle. This is tested by the following specific aims. Aim 1: Determine if SR K or Cl channels carry essential countercurrent during SR Ca release. Aim 2: Determine if SR K or Cl channels provide vital resting SR ion re-equilibration pathways. Aim 3: Establish the mechanism underlying abnormal SR Ca handling in TRIC-A KO muscle. Expected Outcome: Delineate new potential points of SR pathological failure (and/or sites at which SR function can be therapeutically manipulated) by transforming existing "Ca-centric" view of SR function to encompass the multi-ion (Ca, Mg, K, Cl) reality, which governs SR Ca release/uptake.