Devor D C, Forrest J N, Suggs W K, Frizzell R A
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294.
Am J Physiol. 1995 Jan;268(1 Pt 1):C70-9. doi: 10.1152/ajpcell.1995.268.1.C70.
Whole cell and single-channel patch-clamp techniques were used to identify and characterize the Cl- currents responsible for adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion in the rectal gland of the spiny dogfish (Squalus acanthias). During whole cell recordings, in cultured rectal gland cells forskolin (10 microM) and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (400 microM) stimulated a 28-fold increase in Cl- conductance (n = 10). This cAMP-activated conductance pathway had a linear current-voltage (I-V) relationship that was time and voltage independent. Substitution of 235 meq Cl- with I- in the bath inhibited the cAMP-activated current at both positive and negative voltages (64%). Glibenclamide (60 microM) abolished the cAMP-stimulated current, and its effect was irreversible (n = 3). During cell-attached recording, increased cellular cAMP activated single Cl- channels in nine previously quiet patches. These channels had a linear I-V relationship with an average single-channel conductance of 5.1 +/- 0.2 pS (n = 6). Similar properties were observed in excised inside-out patches, permitting further characterization of the single-channel properties. Excised quiescent patches could be activated by the addition of ATP and protein kinase A. Replacing bath Cl- with I- inhibited both inward and outward currents (n = 3). In three inside-out patches, glibenclamide (300 microM) reversibly reduced open probability by 74%, with no effect on single-channel current amplitude. Similar results were obtained in four outside-out recordings. These results suggest that increased cellular cAMP in dogfish rectal gland activates a small linear Cl- channel that resembles human cystic fibrosis transmembrane conductance regulator in its biophysical and pharmacological properties.
采用全细胞和单通道膜片钳技术来识别和表征负责在棘鲨（Squalus acanthias）直肠腺中由3',5'-环磷酸腺苷（cAMP）介导的氯离子分泌的氯离子电流。在全细胞记录过程中，在培养的直肠腺细胞中，福斯高林（10微摩尔）和8-（4-氯苯硫基）-3',5'-环磷酸腺苷（400微摩尔）刺激氯离子电导增加了28倍（n = 10）。这种cAMP激活的电导途径具有与时间和电压无关的线性电流-电压（I-V）关系。用碘离子替代浴液中的235毫当量氯离子，在正电压和负电压下均抑制了cAMP激活的电流（64%）。格列本脲（60微摩尔）消除了cAMP刺激的电流，且其作用是不可逆的（n = 3）。在细胞贴附式记录过程中，细胞内cAMP增加激活了9个先前安静的膜片中的单个氯离子通道。这些通道具有线性I-V关系，平均单通道电导为5.1±0.2皮安（n = 6）。在切除的内面向外膜片中观察到了类似的特性，从而能够进一步表征单通道特性。切除的静止膜片可通过添加ATP和蛋白激酶A来激活。用碘离子替代浴液中的氯离子可抑制内向和外向电流（n = 3）。在3个内面向外膜片中，格列本脲（300微摩尔）使开放概率可逆地降低了74%，对单通道电流幅度无影响。在4个外面向外记录中也获得了类似结果。这些结果表明，棘鲨直肠腺中细胞内cAMP增加激活了一种小的线性氯离子通道，其生物物理和药理学特性类似于人类囊性纤维化跨膜电导调节蛋白。
