Modulation of the pharmacology of Ca2+-activated Cl- channels of pulmonary artery smooth muscle cells and the molecular candidate TMEM16A by phosphorylation.

摘要

The first two studies presented in this dissertation tested the hypothesis that the state of ClCa channel phosphorylation affects their pharmacology. The third study explored whether TMEM16A may be a valid molecular candidate for the native ClCa channel of pulmonary artery myocytes and in particular whether this protein is similarly altered by phosphorylation and classical Cl- channel blockers.The Ca2+-activated Cl- channel (Cl Ca) blocker niflumic acid (NFA) produces a paradoxical dual effect on ICl(Ca), causing stimulation or inhibition at potentials below or above 0 mV, respectively. We first tested whether the effects of NFA on ICl(Ca) are modulated by phosphorylation. Eliciting ICl(Ca) with 500 nM free internal Ca2+ in rabbit pulmonary artery myocytes, the channel's state of phosphorylation was altered by cell dialysis with either 5 mM ATP, or 0 ATP with or without the CaMKII inhibitor KN-93 (10 muM). We found dephosphorylation to enhance the ability of 100 muM NFA to inhibit ICl(Ca), an effect attributable to a large negative shift in the voltage-dependence of block. Channel inhibition was converted to stimulation at potentials -50 mV, &sim70 mV more negative than cells dialyzed with 5 mM ATP. NFA dose-dependently blocked ICl(Ca) in the range of 100 nM to 250 muM in cells dialyzed with 0 ATP and KN-93, which contrasted with the significant stimulation induced by 100 nM, which converted to block at concentrations > 1 muM when cells were dialyzed with 5 mM ATP. Based on these data and the observations that phosphorylation results in state-dependent block and that NFA preferentially interacts with the open channel, we propose two binding sites for NFA an inhibitory site residing near or partially in the channel pare, and a stimulatory site removed from the pore.We then tested the same hypothesis with anthracene-9-carboxylic acid (A9C), a Cl- channel block chemically dissimilar to NFA that like NFA, has a unique dual effect on ICl(Ca), both inhibiting and stimulating the channels depending on voltage and the state of gating. The whole-cell patch clamp technique was used to record ICl(Ca) in rabbit pulmonary artery smooth muscle cells, where currents were generated by 500 nM free internal Ca2+. Again, the state of phosphorylation was altered by cell dialysis with either 5 mM ATP or an ATP-free pipette solution. Similar to NFA, dephosphorylation enhanced the ability of A9C to inhibit I Cl(Ca) in a concentration-dependent manner, with a negative shift in the voltage-dependence of block. Stimulation of ICl(Ca) tail current by 500 muM A9C at -80 mV was enhanced in cells dialyzed with 5 mM ATP. While the tail current of cells dialyzed with 0 ATP was stimulated following depolarization to +40 mV, the stimulation was abolished following steps to +140 mV. These data again suggest the presence of two drug binding sites. It appears that A9C blocks the open channel and that phosphorylation partially occludes access to a blocking site found at the mouth or within the pore. Minimizing phosphorylation resulted in decreased stimulation by A9C of the ICl(Ca) tail current when compared to cells in which phosphorylation was supported, suggesting a phosphorylation-dependent effect on a stimulatory site distinct from the inhibitory site.The final study presented in this dissertation explored TMEM16A as a possible candidate for the native ClCa channel of pulmonary artery myocytes. RT-PCR and immunocytochemistry identified TMEM16A expression in rat and mouse pulmonary artery. HEK293 cells over-expressing TMEM16A displayed large Cl- currents when dialyzed with 500 nM free Ca 2+. These currents displayed similar time- and voltage dependence to native ICl(Ca) of rabbit pulmonary artery smooth muscle cells. Currents ran down, although not to the extent of ICl(Ca) of the native rabbit pulmonary myocytes. The TMEM16A-dependent currents were sensitive to block by NFA and A9C, but demonstrated stimulation by A9C dissimilar from the native current. A mutation of Threonine 610 to an Alanine -- a putative CaMKII phosphorylation site -- did not affect rundown, although it did alter TMEM16A pharmacology, increasing block and stimulation by NFA and A9C, respectively. This study indicates that TMEM16A is a strong candidate for the ClCa channels of pulmonary artery smooth muscle. (Abstract shortened by UMI.)