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.

Public Health Relevance

In skeletal muscle, release of calcium from the sarcoplasmic reticulum drives contraction. Malfunction of the calcium release process is implicated in many skeletal muscle diseases. Here, we define mechanisms that couple overall sarcoplasmic reticulum ionic balance and calcium release. These mechanisms are potential points of pathological failure and/or sites that could be used to manipulate calcium release for therapeutic benefit.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
4R01AR054098-08
Application #
9017808
Study Section
Skeletal Muscle Biology and Exercise Physiology Study Section (SMEP)
Program Officer
Boyce, Amanda T
Project Start
2006-06-01
Project End
2018-02-28
Budget Start
2016-03-01
Budget End
2017-02-28
Support Year
8
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Rush University Medical Center
Department
Physiology
Type
Schools of Medicine
DUNS #
068610245
City
Chicago
State
IL
Country
United States
Zip Code
60612
Zsolnay, Vilmos; Fill, Michael; Gillespie, Dirk (2018) Sarcoplasmic Reticulum Ca2+ Release Uses a Cascading Network of Intra-SR and Channel Countercurrents. Biophys J 114:462-473
Yan, Jiajie; Zhao, Weiwei; Thomson, Justin K et al. (2018) Stress Signaling JNK2 Crosstalk With CaMKII Underlies Enhanced Atrial Arrhythmogenesis. Circ Res 122:821-835
Berti, Claudio; Zsolnay, Vilmos; Shannon, Thomas R et al. (2017) Sarcoplasmic reticulum Ca2+, Mg2+, K+, and Cl- concentrations adjust quickly as heart rate changes. J Mol Cell Cardiol 103:31-39
Uehara, Akira; Murayama, Takashi; Yasukochi, Midori et al. (2017) Extensive Ca2+ leak through K4750Q cardiac ryanodine receptors caused by cytosolic and luminal Ca2+ hypersensitivity. J Gen Physiol 149:199-218
Manno, Carlo; Figueroa, Lourdes C; Gillespie, Dirk et al. (2017) Calsequestrin depolymerizes when calcium is depleted in the sarcoplasmic reticulum of working muscle. Proc Natl Acad Sci U S A 114:E638-E647
Ramos-Franco, Josefina; Fill, Michael (2016) Approaching ryanodine receptor therapeutics from the calcin angle. J Gen Physiol 147:369-73
Berti, Claudio; Furini, Simone; Gillespie, Dirk (2016) PACO: PArticle COunting Method To Enforce Concentrations in Dynamic Simulations. J Chem Theory Comput 12:925-9
Bovo, Elisa; Mazurek, Stefan R; Fill, Michael et al. (2015) Cytosolic Ca²? buffering determines the intra-SR Ca²? concentration at which cardiac Ca²? sparks terminate. Cell Calcium 58:246-53
Gillespie, Dirk; Xu, Le; Meissner, Gerhard (2014) Selecting ions by size in a calcium channel: the ryanodine receptor case study. Biophys J 107:2263-73
Guo, Tao; Nani, Alma; Shonts, Stephen et al. (2013) Sarcoplasmic reticulum K(+) (TRIC) channel does not carry essential countercurrent during Ca(2+) release. Biophys J 105:1151-60

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