Lee Shin-Rong, Adams Paul J, Yue David T
Calcium Signals Laboratory, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
Departments of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
J Physiol. 2015 Jul 1;593(13):2753-78. doi: 10.1113/JP270091.
CaV 2.1 channels constitute a dominant Ca(2+) entry pathway into brain neurons, triggering downstream Ca(2+) -dependent processes such as neurotransmitter release. CaV 2.1 is itself modulated by Ca(2+) , resulting in activity-dependent enhancement of channel opening termed Ca(2+) -dependent facilitation (CDF). Real-time Ca(2+) imaging and Ca(2+) uncaging here reveal that CDF turns out to be strikingly faster, more Ca(2+) sensitive, and larger than anticipated on previous grounds. Robust resolution of the quantitative profile of CDF enables deduction of a realistic biophysical model for this process. These results suggest that CaV 2.1 CDF would figure most prominently in short-term synaptic plasticity and cerebellar Purkinje cell rhythmicity.
CaV 2.1 (P-type) voltage-gated Ca(2+) channels constitute a major source of neuronal Ca(2+) current, strongly influencing rhythmicity and triggering neurotransmitter release throughout the central nervous system. Fitting with such stature among Ca(2+) entry pathways, CaV 2.1 is itself feedback regulated by intracellular Ca(2+) , acting through calmodulin to facilitate channel opening. The precise neurophysiological role of this calcium-dependent facilitation (CDF) remains uncertain, however, in large measure because the very magnitude, Ca(2+) dependence and kinetics of CDF have resisted quantification by conventional means. Here, we utilize the photo-uncaging of Ca(2+) with CaV 2.1 channels fluxing Li(+) currents, so that voltage-dependent activation of channel gating is no longer conflated with Ca(2+) entry, and CDF is then driven solely by light-induced increases in Ca(2+) . By using this strategy, we now find that CDF can be unexpectedly large, enhancing currents by as much as twofold at physiological voltages. CDF is steeply Ca(2+) dependent, with a Hill coefficient of approximately two, a half-maximal effect reached by nearly 500 nm Ca(2+) , and Ca(2+) on/off kinetics in the order of milliseconds to tens of milliseconds. These properties were established for both native P-type currents in cerebellar Purkinje neurons, as well as their recombinant channel counterparts under heterologous expression. Such features suggest that CDF of CaV 2.1 channels may substantially enhance the regularity of rhythmic firing in cerebellar Purkinje neurons, where regularity is believed crucial for motor coordination. In addition, this degree of extensive CDF would be poised to exert large order-of-magnitude effects on short-term synaptic plasticity via rapid modulation of presynaptic Ca(2+) entry.
CaV 2.1通道是大脑神经元中主要的Ca(2+)内流途径，触发下游依赖Ca(2+)的过程，如神经递质释放。CaV 2.1自身受Ca(2+)调节，导致通道开放的活动依赖性增强，称为Ca(2+)依赖性易化（CDF）。实时Ca(2+)成像和Ca(2+)光解笼实验表明，CDF比之前预期的速度更快、对Ca(2+)更敏感且幅度更大。对CDF定量特征的精确解析有助于推导该过程的真实生物物理模型。这些结果表明，CaV 2.1 CDF在短期突触可塑性和小脑浦肯野细胞节律性中起最主要作用。
CaV 2.1（P型）电压门控Ca(2+)通道是神经元Ca(2+)电流的主要来源，强烈影响节律性并在整个中枢神经系统中触发神经递质释放。鉴于在Ca(2+)内流途径中的这种重要地位，CaV 2.1自身受细胞内Ca(2+)的反馈调节，通过钙调蛋白作用促进通道开放。然而，这种钙依赖性易化（CDF）的确切神经生理作用仍不确定，很大程度上是因为CDF的幅度、Ca(2+)依赖性和动力学一直难以用传统方法进行量化。在此，我们利用Ca(2+)光解笼技术，使CaV 2.1通道通过Li(+)电流，这样通道门控的电压依赖性激活就不再与Ca(2+)内流混淆，CDF仅由光诱导的Ca(2+)增加驱动。通过使用这种策略，我们现在发现CDF可能出乎意料地大，在生理电压下可使电流增强多达两倍。CDF对Ca(2+)高度依赖，希尔系数约为2，近500 nM Ca(2+)时达到半数最大效应，Ca(2+)的开启/关闭动力学在毫秒到几十毫秒的量级。这些特性在小脑浦肯野神经元的天然P型电流以及异源表达下的重组通道中均得到证实。这些特征表明，CaV 2.1通道的CDF可能会显著增强小脑浦肯野神经元节律性放电的规律性，而这种规律性被认为对运动协调至关重要。此外，这种程度的广泛CDF可能会通过快速调节突触前Ca(2+)内流，对短期突触可塑性产生大的数量级影响。
