Regulation by Phosphorylation and Functional Significance in Pulmonary Hypertension of Calcium-Activated Chloride/TMEM16A Channels

Calcium-activated chloride channels (ClCa) 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 (ICl(Ca)) are down-regulated by Calcium-calmodulindependent protein kinase II (CaMKII) and up-regulated by the Calcium-dependent protein phosphatase Calcineurin (CaN). In our studies, we tested the hypothesis that ClCa channels in rabbit PASMCs can also be up-regulated by at least one Ca2+-independent serine/threonine protein phosphatase, PP1/PP2A. Additionally, we attempted to ascertain whether ICl(Ca) induced by TMEM16A, the molecular ClCa channel candidate, could be modulated by phosphotransferase activity in a manner similar to the "classical" vascular smooth muscle ICl(Ca) 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/ClCa channels in PH using the monocrotaline (MCT)-induced PH model in the rat. The first part of this dissertation examined the possible regulation of ICl(Ca) in rabbit PASMCs by the Ca2+-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 (RTPCR). Western blot and immunofluorescence experiments also confirmed the presence of all three PP1 isoforms and PP2A. To examine the regulation of ICl(Ca) 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 ii NIPP-1 (100 pM) potently antagonized the recovery of ICl(Ca) 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 ICl(Ca) 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 ICl(Ca) induced by exogenous CaN Aα (0.5 μM). These results demonstrate that ICl(Ca) 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 ICl(Ca) generated by the recently indentified molecular candidate for the ClCa channel TMEM16A is regulated by kinase and phosphatase activity in a similar manner to the "classical" ICl(Ca) in VSM. The whole-cell patch clamp technique was used once again to record ICl(Ca) elicited by 500 nM free Ca2+ in HEK293 cells overexpressing TMEM16A. Intracellular application of the non-selective PP1/PP2A inhibitor okadaic acid (30 nM) significantly attenuated the TMEM16A-induced ICl(Ca) 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 ICl(Ca) 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 ICl(Ca) 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 iii 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 ICl(Ca) 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 ICl(Ca) overexpressed in HEK293 cells are regulated by CaMKII and PP1/PP2A in a manner that is similar to native ICl(Ca) in PASMCs. Our data also suggest that serine 550 is an important contributor to the regulation of ICl(Ca). The final study examined the potential role of TMEM16A/ClCa 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 ICl(Ca) 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 ICl(Ca) were accompanied by parallel changes in the expression of TMEM16A, a gene recently shown to encode for ClCa 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 Ca2+ channel blocker, and NFA (30 or 100 µM, with or without 10 µM Indomethacin to inhibit cyclooxygenases) or T16AInh-A01 (10 µM), TMEM16A/ClCa channel inhibitor, than that of control animals. In conclusion, augmented ClCa/TMEM16A channel activity is a major contributor to the iv 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/ClCa channels in PASMCs as well as a potential pathophysiological function in pulmonary hypertensive vascular tissues