Sarcoplasmic Reticulum SR K Channel Function

描述（由申请人提供）：肌浆网（SR）钙释放代表了一个大的电荷易位，可以非常迅速（<1 ms）将SR膜电位（Vm）移动到钙平衡电位（ECa），在那里净钙释放将停止(1)。然而，这种情况不会发生，因为在释放过程中同时存在逆流。在之前的资助期内，我们确定了ryanodine受体（RyR）的多离子，低选择性孔介导大的反Mg和K通量，同时释放Ca。因此RyR本身携带所需的大部分逆流。这使得SR - K和Cl通道的生理作用尚不清楚。锶钾和氯通道在释放过程中可能携带少量重要的逆流元素，但它们也可能为释放后反离子在锶中重新平衡提供重要途径。事实上，我们对锶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锶钙处理的重要性，它们可能的病理贡献，或者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亚型（A和B）的消融都是胚胎致命的。仅仅消融trica（骨骼肌中的主要形式）会产生明显的SR Ca处理异常（异常的局部Ca释放事件和SR Ca过载；（3-5））。trica - KO肌肉不仅证明了SR Ca处理和SR离子平衡之间存在机制联系，而且提供了一个独特的机会来定义它。在这里，我们将使用trica - KO肌肉来定义复杂的时空Ca， Mg， K和Cl通量如何通过RyR， SR K和SR Cl通道控制SR离子（电压）平衡并调节骨骼肌中SR Ca的处理。这里测试的假设是：在单个骨骼肌SR - Ca释放事件中，SR - K （TRIC）和SR - Cl通道不携带必要的逆流，而是提供关键的SR离子再平衡途径，确保SR离子平衡，SR Vm在释放事件之间返回静息值。再平衡缺陷，而不是缺少逆流，解释了在trici - ko骨骼肌中观察到的SR Ca处理缺陷。这可以通过以下具体目标进行测试。目标1：确定在SR - Ca释放过程中SR - K或Cl通道是否携带必要的逆流。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trica - KO肌异常SR - Ca处理的机制。预期结果：通过将现有的SR功能“以Ca为中心”的观点转变为包含控制SR Ca释放/摄取的多离子（Ca, Mg， K, Cl）现实，描绘SR病理失败的新潜在点（和/或SR功能可以治疗操纵的位置）。

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.