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.
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