Regulation by Phosphorylation and Functional Significance in Pulmonary Hypertension of Calcium-Activated Chloride/TMEM16A Channels
AuthorAyon, Ramon Jose
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Calcium-activated chloride channels (Cl<sub>Ca</sub>) play an important role in numerous physiological processes including cardiac and neuronal excitability, transepithelial fluid transport, and regulation of vascular smooth muscle tone. In arterial smooth muscle cells, Calcium-activated Chloride currents (I<sub>Cl(Ca)</sub>) are down-regulated by Calcium-calmodulin-dependent protein kinase II (CaMKII) and up-regulated by the Calcium-dependent protein phosphatase Calcineurin (CaN). In our studies, we tested the hypothesis that Cl<sub>Ca</sub> channels in rabbit PASMCs can also be up-regulated by at least one Ca<super>2+</super>-independent serine/threonine protein phosphatase, PP1/PP2A. Additionally, we attempted to ascertain whether I<sub>Cl(Ca)</sub> induced by TMEM16A, the molecular Cl<sub>Ca</sub> channel candidate, could be modulated by phosphotransferase activity in a manner similar to the "classical" vascular smooth muscle I<sub>Cl(Ca)</sub> and if it is, which amino acid residue(s) serve as substrate for kinase activity. The final study in this dissertation sought to examine the potential role of TMEM16A/Cl<sub>Ca</sub> channels in PH using the monocrotaline (MCT)-induced PH model in the rat. The first part of this dissertation examined the possible regulation of I<sub>Cl(Ca)</sub> in rabbit PASMCs by the Ca<super>2+</super>-independent protein phosphatases PP1 and/or PP2A. We detected endogenous expression of PP1α, PP1β/δ, PP1γ, PP2Aα, PP2Aβ, PP2Bα (calcineurin (CaN) Aα), and PP2Bβ (CaN Aβ) but not PP2Bγ (CaN Aγ) in rabbit PA using reverse transcriptase PCR (RT-PCR). Western blot and immunofluorescence experiments also confirmed the presence of all three PP1 isoforms and PP2A. To examine the regulation of I<sub>Cl(Ca)</sub> by these phosphatases in PASMCs, we used the conventional whole-cell patch clamp technique. Intracellular dialysis with a peptide inhibitor of calcineurin (CaN-AIP), the non-selective PP1/PP2A inhibitors okadaic acid (0.5, 10, or 30 nM), calyculin A (10 nM), or cantharidin (100 nM), and the selective PP1 inhibitor NIPP-1 (100 pM) potently antagonized the recovery of I<sub>Cl(Ca)</sub> in cells dialyzed with no ATP, whereas the PP2A-selective antagonist fostriecin (30 nM or 150 nM) was ineffective. The combined application of okadaic acid (10 nM) and CaN-autoinhibitory peptide (50 μM) did not potentiate the response of I<sub>Cl(Ca)</sub> in 0 ATP produced by maximally inhibiting CaN or PP1/PP2A alone. Consistent with the non-additive effects of either classes of phosphatases, the PP1 inhibitor NIPP-1 (100 pM) antagonized the recovery of I<sub>Cl(Ca)</sub> induced by exogenous CaN Aα (0.5 μM). These results demonstrate that I<sub>Cl(Ca)</sub> in PA myocytes is regulated by CaN and PP1 and/or PP2A. Our data also suggest the existence of a functional link between these two classes of phosphatases. We next examined whether I<sub>Cl(Ca)</sub> generated by the recently indentified molecular candidate for the Cl<sub>Ca</sub> channel TMEM16A is regulated by kinase and phosphatase activity in a similar manner to the "classical" I<sub>Cl(Ca)</sub> in VSM. The whole-cell patch clamp technique was used once again to record I<sub>Cl(Ca)</sub> elicited by 500 nM free Ca<super>2+</super> in HEK293 cells overexpressing TMEM16A. Intracellular application of the non-selective PP1/PP2A inhibitor okadaic acid (30 nM) significantly attenuated the TMEM16A-induced I<sub>Cl(Ca)</sub> in cells dialyzed with no ATP. Immunocytochemical experiments confirmed the presence of PP1α, PP1β/δ, PP1γ as well as PP2A in HEK293 cells. The potential modulation of TMEM16A-induced I<sub>Cl(Ca)</sub> by CaMKII was tested using two CaMKII specific inhibitors, KN-93 (10 µM) and autocamtide-2-related inhibitory peptide (ARIP; 5 µM). Intracellular dialysis of these CaMKII inhibitors lead to a significant attenuation of the rundown of TMEM16A I<sub>Cl(Ca)</sub> in the presence of 5 mM ATP. Immunocytochemical experiments verified the presence of CaMKII at the protein level in HEK293 cells, while RT-PCR studies identified transcripts for CaMKIIα, CaMKIIβ, CaMKIIγ and CaMKIIδ in HEK293 cells. Mutating a threonine residue at position 623, a putative CaMKII phosphorylation site, to an alanine did not affect rundown, however mutation of a serine to an alanine at position 550, another CaMKII phosphorylation site, partially attenuated I<sub>Cl(Ca)</sub> rundown. The current density of TMEM16A S550A mutants was significantly reduced in HEK293 cells, however it was not associated with a change in voltage sensitivity. These data suggest that TMEM16A-induced I<sub>Cl(Ca)</sub> overexpressed in HEK293 cells are regulated by CaMKII and PP1/PP2A in a manner that is similar to native I<sub>Cl(Ca)</sub> in PASMCs. Our data also suggest that serine 550 is an important contributor to the regulation of I<sub>Cl(Ca)</sub>. The final study examined the potential role of TMEM16A/Cl<sub>Ca</sub> channels in PH using the MCT-induced PH model in the rat. After three weeks post-injection with a single dose of MCT (50 mg/kg IP), the animals developed right ventricular hypertrophy (confirmed by heart weight measurements and 2D-echocardiography) and changes in pulmonary arterial flow (confirmed by pulse-waved Doppler imaging) that were consistent with increased pulmonary arterial pressure and PH. Whole-cell patch clamp experiments revealed a marked increase in niflumic acid (NFA)-sensitive I<sub>Cl(Ca)</sub> density in PASMCs from large conduit and small intralobar pulmonary arteries of MCT-treated rats versus aged-matched saline-injected controls. Quantitative RT-PCR and Western blot analysis revealed that the alterations in I<sub>Cl(Ca)</sub> were accompanied by parallel changes in the expression of TMEM16A, a gene recently shown to encode for Cl<sub>Ca</sub> channels. Finally, the contraction in response to serotonin (5-HT) of conduit and intralobar pulmonary arteries from MCT-treated rats exhibited greater sensitivity to nifedipine (1 µM), an L-type Ca<super>2+</super> channel blocker, and NFA (30 or 100 µM, with or without 10 µM Indomethacin to inhibit cyclooxygenases) or T16A<sub>Inh</sub>-A01 (10 µM), TMEM16A/Cl<sub>Ca</sub> channel inhibitor, than that of control animals. In conclusion, augmented Cl<sub>Ca</sub>/TMEM16A channel activity is a major contributor to the changes in electromechanical coupling and perhaps in the arterial remodeling seen of PA in PH. TMEM16A-encoded channels therefore represents a novel therapeutic target in this disease. In summary, the results presented in this dissertation provide new insight into the mechanisms of regulation by phosphorylation of TMEM16A/Cl<sub>Ca</sub> channels in PASMCs as well as a potential pathophysiological function in pulmonary hypertensive vascular tissues.