Intracellular Signaling In Endocrine Cells

在自发放电的垂体细胞中，已经确定了参与spike去极化和复极化的通道。相比之下，我们对控制静息膜电位和动作电位触发的通道知之甚少。在过去的一年里，我们重点研究了两个非选择性阳离子通道在电活动、钙信号传导和激素分泌中的作用：超极化激活和环核苷酸门控（HCN）通道和经典的瞬时受体电位（TRPC）通道。研究了HCN通道在培养大鼠垂体细胞中的作用。定量RT-PCR分析显示，这些细胞中HCN2和HCN3亚基mRNA转录本的表达水平较高，而HCN1和HCN4亚基的表达水平较低。垂体细胞裂解物的Western免疫印迹分析显示HCN2、HCN3和HCN4具有合适分子量的条带。电生理实验显示，HCN电流存在于促性腺细胞、甲状腺细胞、生长细胞和一小部分乳营养细胞中，以及其他未知的垂体细胞类型中。腺苷酸环化酶的刺激和8-Br-cAMP的加入增强了这种电流并使细胞膜去极化，而8-Br-cGMP没有改变这种电流并使细胞膜超极化。抑制基础腺苷酸环化酶活性和刺激磷脂酶C信号通路均可抑制该电流。然而，抑制HCN通道会影响放电频率，但不会消除自发电活动，这表明其他通道对自发起搏活动至关重要。在培养的乳养菌和永生化GH3细胞的进一步实验中，我们发现，用大的有机阳离子替代细胞外钠，而不是阻断电压门控钠流入，导致细胞膜的瞬时超极化，这与自发放电的停止有关。当将细胞夹在-50 mV（接近这些细胞的静息膜电位）时，用有机阳离子取代浴钠导致向外电流，反映了向内保持膜电流的抑制，并表明背景去极化电导的损失。定量RT-PCR分析显示，乳养细胞和GH3细胞中TRPC1 mRNA转录本高表达，TRPC6 mRNA转录本低表达。垂体细胞中TRPC3、TRPC4和TRPC5 mRNA转录物的表达也很低，但GH3细胞中没有。2-APB和SKF-96365是相对选择性的TRPC通道阻滞剂，以浓度依赖性的方式抑制电活动、钙内流和催乳素释放。非选择性阳离子通道抑制剂钆和氟芬那酸也能抑制电活动、钙内流和催乳素释放。这些结果表明，非选择性阳离子通道，可能属于TRPC家族，有助于这些细胞的背景去极化电导和动作电位的发射。我们正在进行的工作也集中在atp门控的P2X2和P2X7受体通道的结构和功能表征，它们在垂体细胞中表达。在与Sherman博士的合作中，我们最近发现P2X2Rs表现出两种相反的激活依赖性变化，即孔隙扩张和孔隙关闭（脱敏），其过程尚不完全清楚。为了解决这个问题，并阐明钙和c端结构域在门控中的作用，我们结合了生物物理和数学方法。该受体在ATP的2-6秒内对n -甲基- d -葡萄糖胺具有导电性。然而，孔隙扩张伴随着总电导的减少而不是增加，这在时间上与快速和部分脱敏一致。在持续使用激动剂期间，受体继续以钙独立和钙依赖模式脱敏。在全细胞记录中，我们还观察到两种受体的使用依赖性脱敏促进。这种行为被描述ATP结合/解结合和激活/脱敏的16态马尔可夫动力学模型所解释。该模型假设，当两到三个ATP分子结合并进行钙无关脱敏时，神经受体打开，导致总电导降低或孔扩张，导致逆转电位的移动。在含钙介质中，受体脱敏更容易，使用依赖性脱敏可以通过钙依赖性拨动开关来模拟。实验和模型共同提供了扩张P2X2Rs缺乏持续电流增长的基本原理，并表明处于扩张状态的受体在钙存在下也可以脱敏。与Zemkova博士合作，我们还研究了保守的外结构域半胱氨酸残基在P2X7R功能中的作用。在HEK293细胞中表达C119-C168、C129-C152、C135-C162、C216-C226和C260-C269半胱氨酸对单点和双点苏氨酸突变体，并使用全细胞电流记录进行研究。除C119T-P2X7R外，所有突变体都对初始和后续300 M BzATP和ATP的小幅度单相电流有反应，或者实际上没有功能。诱变诱导的功能丧失是由于细胞表面受体表达的减少，正如评估生物素化突变体的水平所揭示的那样。所有双突变体与野生型受体的共表达对受体运输有短暂的抑制作用，或者在C119T/C168T双突变体的情况下，对受体运输有持续的抑制作用。C119T-P2X7R突变体在质膜上表达，功能完全，对BzATP的敏感性略有下降，表明释放的Cys168与另一个残基相互作用挽救了受体的运输。因此，与其他p2xr不同的是，P2X7R的所有二硫键对于适当的受体运输都是必不可少的。我们还研究了Pannexins（一个新发现的三成员蛋白家族）在垂体细胞中的表达模式和作用。去年，我们报道了Pannexin 1 （Panx1）在脑垂体中表达，并提供ATP释放的传递途径。最近的实验表明，除了Panx1的全尺寸异构体（以下简称Panx1a）外，垂体细胞还表达两种新的剪接异构体，称为Panx1c和Panx1d，其形成反映了外显子2和4上存在替代剪接位点。Panx1c缺失Phe108-Gln180序列，P2X1d缺失Val307-Cys426 c端序列。共聚焦显微镜和生物素标记显示，Panx1a在质膜中表达，而Panx1c和Panx1d在作为同质蛋白表达时显示胞质定位。在共表达研究中，我们进一步研究了Panx1a与其两种剪接形式的相互作用，这些短剪接异构体的表达对全尺寸Panx1a通道ATP释放功能的影响，以及它们与P2XRs的关联。三种Panx1同工异构体和Panx2在任何组合中形成同质和异质复合物。这些剪接形式也可以与atp门控的P2X2、P2X3、P2X4和P2X7受体通道物理关联。当与Panx1c或Panx1d共表达时，at -20永生化垂体细胞中panx1a介导的ATP释放减弱。这些结果表明，Panx1c和Panx1d可能作为显性负效应因子，通过异质通道的形成来调节Panx1a的功能。Panx1表达和关联的复杂模式也可以定义这些通道在共表达两种蛋白的细胞类型中的p2x依赖性作用。

Channels contributing to the spike depolarization and repolarization in spontaneously firing pituitary cells have been identified. In contrast, very little is known about channels controlling resting membrane potential and initiation of firing of action potentials. During the last year, we focused on the role of two non-selective cation channels in electrical activity, calcium signaling, and hormone secretion: hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels and classic transient receptor potential (TRPC) channels. The role of HCN channels was studied in cultured rat pituitary cells. Quantitative RT-PCR analysis showed higher level of expression of mRNA transcripts for HCN2 and HCN3 subunits and lower expression of HCN1 and HCN4 subunits in these cells. Western immunoblot analysis of lysates from pituitary cells showed bands with appropriate molecular weights for HCN2, HCN3 and HCN4. Electrophysiological experiments showed the presence of HCN current in gonadotrophs, thyrotrophs, somatotrophs and a fraction of lactotrophs, as well as in other unidentified pituitary cell types. Stimulation of adenylyl cyclase and addition of 8-Br-cAMP enhanced this current and depolarized the cell membrane, whereas 8-Br-cGMP did not alter the current and hyperpolarized the cell membrane. Both inhibition of basal adenylyl cyclase activity and stimulation of phospholipase C signaling pathway inhibited this current. However, inhibition of HCN channels affected the frequency of firing but did not abolish spontaneous electrical activity, indicating that other channels are critical for spontaneous pacemaking activity. In further experiments with cultured lactotrophs and immortalized GH3 cells, we found that replacement of extracellular sodium with large organic cations, but not blockade of voltage-gated sodium influx, led to an instantaneous hyperpolarization of cell membranes that was associated with a cessation of spontaneous firing. When cells were clamped at -50 mV, which was close to the resting membrane potential in these cells, replacement of bath sodium with organic cations resulted in an outward-like current, reflecting an inhibition of the inward holding membrane current and indicating loss of a background-depolarizing conductance. Quantitative RT-PCR analysis revealed the high expression of mRNA transcripts for TRPC1 and much lower expression of TRPC6 in both lactotrophs and GH3 cells. Very low expression of TRPC3, TRPC4, and TRPC5 mRNA transcripts were also present in pituitary but not GH3 cells. 2-APB and SKF-96365, relatively selective blockers of TRPC channels, inhibited electrical activity, calcium influx and prolactin release in a concentration-dependent manner. Gadolinium, and flufenamic acid, inhibitors of non-selective cation channels, also inhibited electrical activity, calcium influx and prolactin release. These results indicate that nonselective cation channels, presumably belonging to the TRPC family, contribute to the background depolarizing conductance and firing of action potentials in these cells. Our ongoing work is also focused on structural and functional characterization of ATP-gated P2X2 and P2X7 receptor-channels, which are expressed in pituitary cells. In collaboration with Dr. Sherman, we recently found that P2X2Rs exhibit two opposite activation-dependent changes, pore dilation and pore closing (desensitization), through a process that is incompletely understood. To address this issue and to clarify the roles of calcium and the C-terminal domain in gating, we combined biophysical and mathematical approaches. This receptor developed conductivity for N-methyl-D-glucamine within 2-6 s of ATP application. However, pore dilation was accompanied with a decrease rather than an increase in the total conductance, which temporally coincided with rapid and partial desensitization. During sustained agonist application, receptors continued to desensitize in calcium-independent and calcium-dependent modes. In whole-cell recording, we also observed use-dependent facilitation of desensitization of both receptors. Such behavior was accounted for by a 16-state Markov kinetic model describing ATP binding/unbinding and activation/desensitization. The model assumes that nave receptors open when two to three ATP molecules bind and undergo calcium-independent desensitization, causing a decrease in the total conductance, or pore dilation, causing a shift in the reversal potential. In calcium-containing media, receptor desensitization is facilitated and the use-dependent desensitization can be modeled by a calcium-dependent toggle switch. The experiments and the model together provide a rationale for the lack of sustained current growth in dilating P2X2Rs and show that receptors in the dilated state can also desensitize in the presence of calcium. In collaboration with Dr. Zemkova, we also studied the role of conserved ectodomain cysteine residues in P2X7R function. Single- and double-point threonine mutants of C119-C168, C129-C152, C135-C162, C216-C226, and C260-C269 cysteine pairs were expressed in HEK293 cells and studied using whole-cell current recording. All mutants other than C119T-P2X7R responded to initial and subsequent application of 300 M BzATP and ATP with small amplitude monophasic currents or were practically non-functional. The mutagenesis-induced loss of function was due to decreased cell-surface receptor expression, as revealed by assessing levels of biotinylated mutants. Coexpression of all double mutants with the wild type receptor had a transient or, in the case of C119T/C168T double mutant, sustained inhibitory effect on receptor trafficking. The C119T-P2X7R mutant was expressed on the plasma membrane and was fully functional with a slight decrease in the sensitivity for BzATP, indicating that interaction of liberated Cys168 with another residue rescues the trafficking of receptor. Thus, in contrast to other P2XRs, all disulfide bonds of P2X7R are individually essential for the proper receptor trafficking. We also studied the expression pattern and role of Pannexins, a newly discovered three-member family of proteins, in pituitary cells. The last year, we reported that Pannexin 1 (Panx1) is expressed in the pituitary gland and provides a pathway for delivery of ATP release. Recent experiments revealed that, in addition to the full size isoform of Panx1, hereafter referred to as Panx1a, pituitary cells also express two novel splice isoforms, termed Panx1c and Panx1d, which formation reflects the existence of alternative splicing sites in exons 2 and 4. Panx1c is lacking the Phe108-Gln180 sequence and P2X1d is missing the Val307-Cys426 C-terminal end sequence. Confocal microscopy and biotin labeling revealed that Panx1a is expressed in the plasma membrane, whereas Panx1c and Panx1d show the cytoplasmic localization when expressed as homomeric proteins. In co-expression studies, we further investigated the interactions of Panx1a with its two splice forms, the effect of expression of these short splice isoforms on the ATP release functions of full-size Panx1a channels, and their association with P2XRs. The three Panx1 isoforms and Panx2 form homomeric and heteromeric complexes in any combination. These splice forms can also physically associate with ATP-gated P2X2, P2X3, P2X4, and P2X7 receptor channels. The Panx1a-mediated ATP release in AtT-20 immortalized pituitary cells is attenuated when co-expressed with Panx1c or Panx1d. These results suggest that Panx1c and Panx1d may serve as dominant-negative effectors to modulate the functions of Panx1a through formation of heteromeric channels. The complex patterns of Panx1 expression and association could also define the P2X-dependent roles of these channels in cell types co-expressing both proteins.