Inchauspe Carlota González, Forsythe Ian D, Uchitel Osvaldo D
Instituto de Fisiología, Biología Molecular y Neurociencias, CONICET, Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina.
J Physiol. 2007 Nov 1;584(Pt 3):835-51. doi: 10.1113/jphysiol.2007.139683. Epub 2007 Sep 6.
P/Q-type and N-type calcium channels mediate transmitter release at rapidly transmitting central synapses, but the reasons for the specific expression of one or the other in each particular synapse are not known. Using whole-cell patch clamping from in vitro slices of the auditory brainstem we have examined presynaptic calcium currents (I(pCa)) and glutamatergic excitatory postsynaptic currents (EPSCs) at the calyx of Held synapse from transgenic mice in which the alpha(1A) pore-forming subunit of the P/Q-type Ca(2+) channels is ablated (KO). The power relationship between Ca(2+) influx and quantal output was studied by varying the number of Ca(2+) channels engaged in triggering release. Our results have shown that more overlapping Ca(2+) channel domains are required to trigger exocytosis when N-type replace P/Q-type calcium channels suggesting that P/Q type Ca(2+) channels are more tightly coupled to synaptic vesicles than N-type channels, a hypothesis that is verified by the decrease in EPSC amplitudes in KO synapses when the slow Ca(2+) buffer EGTA-AM was introduced into presynaptic calyces. Significant alterations in short-term synaptic plasticity were observed. Repetitive stimulation at high frequency generates short-term depression (STD) of EPSCs, which is not caused by presynaptic Ca(2+) current inactivation neither in WT or KO synapses. Recovery after STD is much slower in the KO than in the WT mice. Synapses from KO mice exhibit reduced or no EPSC paired-pulse facilitation and absence of facilitation in their presynaptic N-type Ca(2+) currents. Simultaneous pre- and postsynaptic double patch recordings indicate that presynaptic Ca(2+) current facilitation is the main determinant of facilitation of transmitter release. Finally, KO synapses reveal a stronger modulation of transmitter release by presynaptic GTP-binding protein-coupled receptors (gamma-aminobutyric acid type B receptors, GABA(B), and adenosine). In contrast, metabotropic glutamate receptors (mGluRs) are not functional at the synapses of these mice. These experiments reinforce the idea that presynaptic Ca(2+) channels expression may be tuned for speed and modulatory control through differential subtype expression.
P/Q型和N型钙通道介导快速传递的中枢突触处的递质释放，但每个特定突触中特异性表达其中一种通道的原因尚不清楚。我们使用听觉脑干体外切片的全细胞膜片钳技术，检测了来自P/Q型Ca(2+)通道的α(1A)孔形成亚基被敲除（KO）的转基因小鼠的Held壶腹突触处的突触前钙电流（I(pCa)）和谷氨酸能兴奋性突触后电流（EPSCs）。通过改变参与触发释放的Ca(2+)通道数量，研究了Ca(2+)内流与量子输出之间的幂关系。我们的结果表明，当N型钙通道取代P/Q型钙通道时，触发胞吐作用需要更多重叠的Ca(2+)通道结构域，这表明P/Q型Ca(2+)通道比N型通道与突触小泡的耦合更紧密，当将慢Ca(2+)缓冲剂EGTA-AM引入突触前壶腹时，KO突触中EPSC振幅的降低验证了这一假设。观察到短期突触可塑性有显著改变。高频重复刺激会导致EPSCs的短期抑制（STD），这在野生型或KO突触中均不是由突触前Ca(2+)电流失活引起的。KO小鼠中STD后的恢复比野生型小鼠慢得多。KO小鼠的突触表现出EPSC双脉冲易化降低或无易化，且其突触前N型Ca(2+)电流无易化。同时进行突触前和突触后双膜片钳记录表明，突触前Ca(2+)电流易化是递质释放易化的主要决定因素。最后，KO突触显示出突触前GTP结合蛋白偶联受体（γ-氨基丁酸B型受体，GABA(B)，和腺苷）对递质释放的更强调节作用。相比之下，代谢型谷氨酸受体（mGluRs）在这些小鼠的突触中不起作用。这些实验强化了这样一种观点，即突触前Ca(2+)通道的表达可能通过不同亚型的表达来调节速度和调节控制。
