Kanaporis Giedrius, Mese Gulistan, Valiuniene Laima, White Thomas W, Brink Peter R, Valiunas Virginijus
Department of Physiology and Biophysics, Stony Brook University, Stony Brook, NY 11794, USA.
J Gen Physiol. 2008 Apr;131(4):293-305. doi: 10.1085/jgp.200709934.
Gap junction channels exhibit connexin dependent biophysical properties, including selective intercellular passage of larger solutes, such as second messengers and siRNA. Here, we report the determination of cyclic nucleotide (cAMP) permeability through gap junction channels composed of Cx43, Cx40, or Cx26 using simultaneous measurements of junctional conductance and intercellular transfer of cAMP. For cAMP detection the recipient cells were transfected with a reporter gene, the cyclic nucleotide-modulated channel from sea urchin sperm (SpIH). cAMP was introduced via patch pipette into the cell of the pair that did not express SpIH. SpIH-derived currents (I(h)) were recorded from the other cell of a pair that expressed SpIH. cAMP diffusion through gap junction channels to the neighboring SpIH-transfected cell resulted in a five to sixfold increase in I(h) current over time. Cyclic AMP transfer was observed for homotypic Cx43 channels over a wide range of conductances. However, homotypic Cx40 and homotypic Cx26 exhibited reduced cAMP permeability in comparison to Cx43. The cAMP/K(+) permeability ratios were 0.18, 0.027, and 0.018 for Cx43, Cx26, and Cx40, respectively. Cx43 channels were approximately 10 to 7 times more permeable to cAMP than Cx40 or Cx26 (Cx43 > Cx26 > or = Cx40), suggesting that these channels have distinctly different selectivity for negatively charged larger solutes involved in metabolic/biochemical coupling. These data suggest that Cx43 permeability to cAMP results in a rapid delivery of cAMP from cell to cell in sufficient quantity before degradation by phosphodiesterase to trigger relevant intracellular responses. The data also suggest that the reduced permeability of Cx26 and Cx40 might compromise their ability to deliver cAMP rapidly enough to cause functional changes in a recipient cell.
间隙连接通道具有依赖连接蛋白的生物物理特性，包括允许较大溶质（如第二信使和小干扰RNA）在细胞间选择性通过。在此，我们报告了通过同时测量连接电导和细胞间cAMP转移，测定由Cx43、Cx40或Cx26组成的间隙连接通道对环核苷酸（cAMP）的通透性。为了检测cAMP，受体细胞用一个报告基因——海胆精子的环核苷酸调节通道（SpIH）进行转染。通过膜片钳吸管将cAMP引入未表达SpIH的细胞对中的一个细胞。从表达SpIH的细胞对中的另一个细胞记录SpIH衍生电流（I(h)）。cAMP通过间隙连接通道扩散到相邻的SpIH转染细胞，导致I(h)电流随时间增加五到六倍。在很宽的电导范围内，同型Cx43通道都观察到了cAMP转移。然而，与Cx43相比，同型Cx40和同型Cx26的cAMP通透性降低。Cx43、Cx26和Cx40的cAMP/K(+)通透性比值分别为0.18、0.027和0.018。Cx43通道对cAMP的通透性比Cx40或Cx26高约10至7倍（Cx43 > Cx26 >或 = Cx40），这表明这些通道对参与代谢/生化偶联的带负电荷的较大溶质具有明显不同的选择性。这些数据表明，Cx43对cAMP的通透性导致cAMP在细胞间迅速大量传递，在被磷酸二酯酶降解之前足以触发相关的细胞内反应。数据还表明，Cx26和Cx40通透性的降低可能会损害它们将cAMP迅速传递到足以引起受体细胞功能变化的能力。
