Hotchkiss Brain and Libin Cardiovascular Research Institutes and Department of Physiology and Pharmacology, University of Calgary, AB, T2N 4N1, Canada.
J Cell Sci. 2013 Jul 1;126(Pt 13):2944-54. doi: 10.1242/jcs.128363. Epub 2013 Apr 23.
Recent investigations have identified that T-type Ca(2+) channels (CaV3.x) are expressed in rat cerebral arterial smooth muscle. In the study reported here, we isolated the T-type conductance, differentiated the current into the CaV3.1/CaV3.2 subtypes and determined whether they are subject to protein kinase regulation. Using patch clamp electrophysiology, whole-cell Ba(2+) current was monitored and initially subdivided into nifedipine-sensitive and -insensitive components. The latter conductance was abolished by T-type Ca(2+) channel blockers and was faster with leftward shifted activation/inactivation properties, reminiscent of a T-type channel. Approximately 60% of this T-type conductance was blocked by 50 µM Ni(2+), a concentration that selectively interferes with CaV3.2 channels. Subsequent work revealed that the whole-cell T-type conductance was subject to protein kinase A (PKA) modulation. Specifically, positive PKA modulators (db-cAMP, forskolin, isoproterenol) suppressed T-type currents and evoked a hyperpolarized shift in steady-state inactivation. Blocking PKA (with KT5720) masked this suppression without altering the basal T-type conductance. A similar effect was observed with stHt31, a peptide inhibitor of A-kinase anchoring proteins. A final set of experiments revealed that PKA-induced suppression targeted the CaV3.2 subtype. In summary, this study revealed that a T-type Ca(2+) channel conductance can be isolated in arterial smooth muscle, and differentiated into CaV3.1 and CaV3.2 components. It also showed that vasodilatory signaling cascades inhibit this conductance by targeting CaV3.2. Such targeting would impact Ca(2+) dynamics and consequent tone regulation in the cerebral circulation.
最近的研究表明 T 型钙通道（CaV3.x）在大鼠脑动脉平滑肌中表达。在本研究中，我们分离了 T 型电流，将电流分为 CaV3.1/CaV3.2 亚型，并确定它们是否受到蛋白激酶的调节。使用膜片钳电生理学技术，监测全细胞膜片钳 Ba(2+)电流，初步将其分为硝苯地平敏感和不敏感成分。后一种电导率被 T 型钙通道阻滞剂消除，激活/失活特性向左移，类似于 T 型通道。大约 60%的这种 T 型电流被 50 µM Ni(2+)阻断，该浓度选择性地干扰 CaV3.2 通道。随后的研究表明，全细胞膜片钳 T 型电流受到蛋白激酶 A (PKA)调节。具体来说，PKA 正向调节剂（db-cAMP、forskolin、异丙肾上腺素）抑制 T 型电流并引起稳态失活的超极化移位。用 KT5720 阻断 PKA 掩盖了这种抑制作用，而不改变基础 T 型电流。A-kinase 锚定蛋白的肽抑制剂 stHt31 也观察到类似的效果。最后一组实验表明，PKA 诱导的抑制作用针对 CaV3.2 亚型。总之，这项研究表明，动脉平滑肌中可以分离出 T 型钙通道电流，并将其分为 CaV3.1 和 CaV3.2 成分。它还表明，血管舒张信号级联通过靶向 CaV3.2 抑制这种电导。这种靶向作用将影响大脑循环中的钙动力学和随后的张力调节。
