Abstract
The present study was undertaken to investigate the effects of specific inhibitors of calmodulin-dependent protein kinase II (CamKII) on macroscopic voltage-dependent K+ current (KV) recorded from rabbit portal vein smooth muscle cells. Inhibition of L-type Ca2+ current facilitation by 1 μM KN-62, a blocker of CamKII, was first demonstrated and provided evidence for functional CamKII activity in this preparation. KN-93, another specific and more potent inhibitor of CamKII in the rat brain, suppressed KVand enhanced the rate of inactivation in a dose-dependent manner, in cells dialyzed with both low (0.1 mM) and high (10 mM) EGTA pipette solution. Prolonged dialysis with 10 μM of a synthetic peptide inhibitor of CamKII (fragment 281–301) had little effect on KV and did not prevent the inhibitory action of KN-93 on the current. The estimated IC50 for inhibiting peak and late currents during 250-ms steps to +60 mV (holding potential = −60 mV) were 2.9 and 0.27 μM, respectively. KN-93 also induced slight shifts of the steady-state activation (−7 mV) and inactivation (−6 mV) curves. KN-62, and KN-92, an inactive analog of KN-93, produced effects similar to those of KN-93. In current clamp experiments, 5 μM KN-93 depolarized the myocytes from a control resting membrane potential of −42.3 ± 2.8 mV to −28.5 ± 1.4 mV, an effect that was partially reversible after washout (−34.4 ± 1.3 mV, n = 6). In conclusion, blockers of CamKII produce nonspecific inhibitory effects on KV that warrant cautious use of these compounds in physiological experiments designed to assess the role of CamKII.
Footnotes
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Send reprint requests to: Normand Leblanc, Ph.D., Research Centre, Montreal Heart Institute, 5000 East Bélanger St., Montréal, Québec, Canada H1T 1C8. E-mail:leblancn{at}alize.ere.umontreal.ca.
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↵1 This work was supported by grants awarded to N.L. from the Heart and Stroke Foundation of Québec, the Medical Research Council of Canada, and funds from the Fonds pour la Formation de Chercheurs et l’Aide à la Recherche (FCAR) and Montreal Heart Institute. N.L. is a Fonds de la Recherche en Santé du Québec Senior Scholar.
- Abbreviations:
- CamKII
- calmodulin-dependent protein kinase II
- HP
- holding potential
- 4-AP
- 4-aminopyridine
- [Ca2+]i
- free intracellular calcium concentration
- DMSO
- dimethyl sulfoxide
- ICa
- L-type calcium current
- Ito
- transient outward K+current
- KATP
- ATP-dependent K+ channels
- KCa
- Ca2+-dependent K+ channels
- KV
- voltage-dependent K+ channels
- PSS
- physiological salt solution
- RMP
- resting membrane potential
- TEA
- tetraethylammonium chloride
- Received November 4, 1998.
- Accepted May 19, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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