Faculty Bibliography

The channel proteins responsible for the cardiac transient outward K+ current (Ito) of human and rodent heart are composed, in part, of pore-forming Kv4.3 or Kv4.2 principal subunits. Recent reports implicate K+ channel interacting proteins (members of the neuronal Ca2+-binding protein family) as subunits of the Ito channel complex. We reported that another Ca2+-binding protein, frequenin [or neuronal calcium center protein-1 (NCS-1)], also functions as a Kv4 auxiliary subunit in the brain. By examining cardiac expression of NCS-1, the aim of this study was to examine the potential physiologic relevance of this protein as an additional regulator of cardiac Ito. Immunoblot analysis demonstrates NCS-1 protein to be expressed in adult mouse ventricle at levels comparable to that found in some brain regions. Cardiac NCS-1 protein expression levels are much higher in fetal and neonatal mouse hearts when compared with the adult. Immunocytochemical analysis of isolated neonatal mouse ventricular myocytes demonstrates co-localization of NCS-1 and Kv4.2 proteins at the sarcolemma. Given its high levels of expression in the heart, NCS-1 should be considered an important potential Kv4 regulatory subunit, particularly in the immature heart

Phosphatidylinositol 4,5-bisphosphate (PIP2) stimulates ATP-sensitive K+ (K(ATP)) channel activity. Because phospholipase C (PLC) hydrolyzes membrane-bound PIP2, which in turn may potentially decrease K(ATP) channel activity, we investigated the effects of the alpha1-adrenoceptor-G(q)-PLC signal transduction axis on pinacidil-activated K(ATP) channel activity in adult rat and neonatal mouse ventricular myocytes. The alpha1-adrenoceptor agonist methoxamine (MTX) reversibly inhibited the pinacidil-activated K(ATP) current in a concentration-dependent manner (IC50 20.9+/-6.6 micromol/L). This inhibition did not occur when the specific alpha1-adrenoceptor antagonist, prazosin, was present. An involvement of G proteins is suggested by the ability of GDPbetaS to prevent this response. Blockade of PLC by U-73122 (2 micromol/L) or neomycin (2 mmol/L) attenuated the MTX-induced inhibition of K(ATP) channel activity. In contrast, the MTX response was unaffected by protein kinase C inhibition or stimulation by H-7 (100 micro mol/L) or phorbol 12,13-didecanoate. The MTX-induced inhibition became irreversible in the presence of wortmannin (20 micro mol/L), an inhibitor of phosphatidylinositol-4 kinase, which is expected to prevent membrane PIP2 replenishment. In excised inside-out patch membranes, pinacidil induced a significantly rightward shift of ATP sensitivity of the channel. This phenomenon was reversed by pretreatment of myocytes with MTX. Direct visualization of PIP2 subcellular distribution using a PLCdelta pleckstrin homology domain-green fluorescent protein fusion constructs revealed reversible translocation of green fluorescent protein fluorescence from the membrane to the cytosol after alpha1-adrenoceptor stimulation. Our data demonstrate that alpha1-adrenoceptor stimulation reduces the membrane PIP2 level, which in turn inhibits pinacidil-activated K(ATP) channels