■宽松膜片钳技术首先被开发并用于天然两栖动物骨骼肌(SkM),提供有用的功能补充传统的锋利的微电极,间隙,或常规贴片电压钳位。它证明了ryanodine受体(RyR)介导的Ca2释放对Na通道(Nav1.4)电流的药理学修饰的反馈作用,在天然鼠SkM中启动激发-收缩耦合。其他RyR和Ca2-ATPase(SERCA)拮抗剂的作用,丹曲林和环吡唑酸(CPA),在这些作用中还涉及背景肾小管-肌浆Ca2结构域。
■我们将松散膜片钳方法扩展到鼠海马脑片coruammonis-1(CA1)锥体神经元的离子电流测量。我们探索了药理学操纵RyR和SERCA介导的细胞内储存Ca2释放和再摄取对Na电流的影响。我们采用了以前应用于天然骨骼肌的方案。这些证明了Ca2介导的对Na通道功能的反馈作用。
■使用去极化15ms持续时间的松散膜片钳步骤来测试电压范围为-40至120mV正的静息膜电位的实验表明,0.5mM咖啡因降低了内向电流幅度,同意之前的SkM调查结果。它还降低了瞬态,但没有延长向外的电流幅度。然而,2mM咖啡因既不影响向内也不影响瞬时向外,但增加了延长的外向电流,与SkM中不断增加的内向电流相反。此外,与之前的SKM发现类似,丹曲林(10μM)和CPA(1μM)给药前均保持内向电流和外向电流不变。然而,丹曲林预处理仍然消除了随后的0.5和2mM咖啡因挑战对内向电流和外向电流的影响。最后,CPA消除了0.5mM咖啡因对内向电流和外向电流的影响,但是有了2mM的咖啡因,内向电流和瞬态外向电流没有变化,但是持续的外向流增加了。
■我们,因此,扩展松片钳夹以建立鼠CA1锥体神经元的药理特性及其与SkM的相似性和对比。这里,诱发,尽管不是背景Ca2+-存储释放影响了Nav和Kv激发,与背景存储Ca2+释放对静息[Ca2+]的贡献较小一致。这种潜在的非规范机制可以调节神经元膜兴奋性或细胞放电速率。
UNASSIGNED: The loose-patch clamp technique was first developed and used in native amphibian skeletal muscle (SkM), offering useful features complementing conventional sharp micro-electrode, gap, or conventional patch voltage clamping. It demonstrated the feedback effects of pharmacological modification of ryanodine receptor (RyR)-mediated Ca2+ release on the Na+ channel (Nav1.4) currents, initiating excitation-contraction coupling in native murine SkM. The effects of the further RyR and Ca2+-ATPase (SERCA) antagonists,
dantrolene and cyclopiazonic acid (CPA), additionally implicated background tubular-sarcoplasmic Ca2+ domains in these actions.
UNASSIGNED: We extend the loose-patch clamp approach to ion current measurements in murine hippocampal brain slice cornu ammonis-1 (CA1) pyramidal neurons. We explored the effects on Na+ currents of pharmacologically manipulating RyR and SERCA-mediated intracellular store Ca2+ release and reuptake. We adopted protocols previously applied to native skeletal muscle. These demonstrated Ca2+-mediated feedback effects on the Na+ channel function.
UNASSIGNED: Experiments applying depolarizing 15 ms duration loose-patch clamp steps to test voltages ranging from -40 to 120 mV positive to the resting membrane potential demonstrated that 0.5 mM caffeine decreased inward current amplitudes, agreeing with the previous SkM findings. It also decreased transient but not prolonged outward current amplitudes. However, 2 mM caffeine affected neither inward nor transient outward but increased prolonged outward currents, in contrast to its increasing inward currents in SkM. Furthermore, similarly and in contrast to previous SkM findings, both
dantrolene (10 μM) and CPA (1 μM) pre-administration left both inward and outward currents unchanged. Nevertheless,
dantrolene pretreatment still abrogated the effects of subsequent 0.5- and 2-mM caffeine challenges on both inward and outward currents. Finally, CPA abrogated the effects of 0.5 mM caffeine on both inward and outward currents, but with 2 mM caffeine, inward and transient outward currents were unchanged, but sustained outward currents increased.
UNASSIGNED: We, thus, extend loose-patch clamping to establish pharmacological properties of murine CA1 pyramidal neurons and their similarities and contrasts with SkM. Here, evoked though not background Ca2+-store release influenced Nav and Kv excitation, consistent with smaller contributions of background store Ca2+ release to resting [Ca2+]. This potential non-canonical mechanism could modulate neuronal membrane excitability or cellular firing rates.