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Regulation of Kv7.5 Potassium Channels by Protein Kinase C in Human Airway Smooth Muscle Cells
Author(s) -
Haick Jennifer M,
Brueggemann Lioubov I,
Byron Kenneth L
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1263.4
Subject(s) - protein kinase c , trachealis muscle , potassium channel , phosphorylation , chemistry , phorbol , endocrinology , medicine , histamine , activator (genetics) , patch clamp , microbiology and biotechnology , biology , receptor , biochemistry , charybdotoxin
Kv7.5 potassium (K + ) channels were recently found to be expressed in airway smooth muscle cells (ASMCs). Bronchoconstrictor agonists inhibit the outward flux of K + through these channels, an effect which has been implicated as a mediator of their bronchoconstrictor actions in human airways. To test the hypothesis that Kv7.5 channels in ASMCs are regulated by bronchoconstrictor agonists via protein kinase C‐ (PKC‐) mediated phosphorylation, FLAG‐tagged human Kv7.5 channels were overexpressed in human trachealis smooth muscle cells (hASMCs). Function of the channels was assessed by measuring outward Kv7.5 currents using whole cell patch clamp electrophysiology. Indirect activation of PKC by the bronchoconstrictor agonist histamine (1 μM) reduced Kv7.5 currents by 34.6 ± 7% at −20 mV (n=6, p<0.05). This effect was abolished upon blockade of the histamine H1 receptor with chlorpheniramine (30 μM). The PKC activator phorbol 12‐myristate 13‐acetate (PMA, 1 nM), but not its inactive analog 4α‐PMA (1 nM), also reduced Kv7.5 currents (75.5 ± 7.6% at −20 mV, n=4, p<0.05). Phosphorylation of the channel protein was assessed by immunoprecipitating the channels (using anti‐FLAG antibodies) and Western blotting with anti‐phosphoserine PKC substrate antibodies. Both histamine and PMA significantly increased Kv7.5 channel phosphorylation and this effect was reduced by the PKC inhibitor Ro‐31‐8220 (1–10 μM). Two putative PKC‐phosphorylation sites on Kv7.5, serine 441 (S441) and threonine 505 (T505), were mutated so that they can no longer be phosphorylated. Neither Kv7.5‐T505A or Kv7.5‐S441A currents were inhibited by histamine (<8% inhibition at −20 mV, n=5, p>0.05, and <14% inhibition at −20 mV, n=7, p>0.05, respectively), however both mutant channels remained sensitive to PMA (58.3 ± 16.8% at −20mV, n=5, p<0.05, and 63 ± 7.5% at −20 mV, n=6, p<0.05, respectively). Despite the insensitivity of Kv7.5‐S441A currents to histamine treatment, Western blot analyses indicated that histamine still induced serine phosphorylation of Kv7.5‐S441A channels. Determining the mechanism by which Kv7.5 channels are regulated in hASMCs will aid in the understanding of how these channels contribute to the regulation of airway diameter and how Kv7 channels in ASMCs may be effectively targeted for treatment of diseases associated with airway obstruction, such as asthma. Support or Funding Information Loyola University Chicago