W-7 modulates K_v4.3: pore block and Ca²⁺-calmodulin inhibition

W-7 inhibits voltage-dependent K+ channels independent of calmodulin activity in rabbit coronary arterial smooth muscle cells

“…For example, W-7 directly blocks human ether a-go-go-related gene (hERG) potassium channels and hKv1.5 channels expressed in human embryonic kidney (HEK) 293 cells (Zhang et al, 2010). W-7 also inhibited the Kv4.3 channels expressed in Xenopus oocytes (Qu et al, 2007). However, studies to date have been restricted to cultured cell lines; no studies have tried to reveal the effect of W-7 on Kv channels in native vascular smooth muscle cells.…”

Closed-state inactivation in Kv4.3 isoforms is differentially modulated by protein kinase C

“…Wild-type (WT) cDNA of rat Kv4.3 (short form) was a gift of Dr. David McKinnon (SUNY Stony Brook, Stony Brook, NY), and its use has been previously described (14,38). Synthetic mRNA was prepared using a mMESSAGE mMACHINE T7 kit (Ambion, Austin, TX ) were prepared by performing PCR-based site-directed mutagenesis as previously described (13,33).…”

“…All protocols were approved by the University at Buffalo State University of New York Institutional Animal Care and Use Committee. Mature female X. laevis (Xenopus One, Ann Arbor, MI) were anesthetized by immersion in a solution of ethyl 3-aminobenzoate methane sulfonate salt (1.5 g/l; Sigma-Aldrich, St Louis, MO), and ovarian lobes were removed through a small incision in the abdominal wall as previously described (37,38). The lobes were placed in a collagenase-containing, Ca 2ϩ -free OR2 solution [in mM: 82.5 NaCl, 2 KCl, 1 MgCl2, 5 HEPES, pH 7.4, with 1-2 mg/ml collagenase (type II; Sigma, St Louis, MO)] to remove the follicular layer.…”

“…The lobes were placed in a collagenase-containing, Ca 2ϩ -free OR2 solution [in mM: 82.5 NaCl, 2 KCl, 1 MgCl2, 5 HEPES, pH 7.4, with 1-2 mg/ml collagenase (type II; Sigma, St Louis, MO)] to remove the follicular layer. The solution containing the oocytes was gently agitated for about 1.5 h, and collagenase activity was then arrested by bovine albumin as previously described (14,38). Defolliculated oocytes (stages V and VI) were then injected with synthetic mRNA (up to 25 ng) using a Nanoject microinjection system (Drummond Scientific, Broomall, PA) and incubated at 18°C for 24 -72 h in an antibiotic-containing Barth's solution [in mM: 88 NaCl, 1 KCl, 2.4 NaHCO 3, 0.82 MgSO4, 0.33 Ca(NO3)2, 0.41 CaCl2, and 10 HEPES, pH 7.4, with 1% antibiotic-antimycotic solution (Invitrogen)].…”

“…Oocytes were voltage-clamped using a two-microelectrode high-performance oocyte clamp amplifier (CA1-B; Dagan, Minneapolis, MN), as described in detail previously (14,38). Microelectrodes were fabricated from 1.5-mm-outer diameter borosilicate glass tubing (TW150F-4; WPI) using a two-stage puller (L/M-3 P-A; Adams & List, Great Neck, NY) filled with 3 M KCl and having resistances of 0.6 -1.5 M⍀.…”