Na+ channel regulation by Ca2+/calmodulin and Ca2+/calmodulin-dependent protein kinase II in guinea-pig ventricular myocytes.

AIMS: Calmodulin (CaM) regulates Na+ channel gating through binding to an IQ-like motif in the C-terminus. Ca2+/CaM-dependent protein kinase II (CaMKII) regulates Ca2+ handling, and chronic overactivity of CaMKII is associated with left ventricular hypertrophy and dysfunction and lethal arrhythmias. However, the acute effects of Ca2+/CaM and CaMKII on cardiac Na+ channels are not fully understood. METHODS AND RESULTS: Purified Na(V)1.5-glutathione-S-transferase fusion peptides were phosphorylated in vitro by CaMKII predominantly on the I-II linker. Whole-cell voltage-clamp was used to measure Na+ current (I(Na)) in isolated guinea-pig ventricular myocytes in the absence or presence of CaM or CaMKII in the pipette solution. CaMKII shifted the voltage dependence of Na+ channel availability by approximately +5 mV, hastened recovery from inactivation, decreased entry into intermediate or slow inactivation, and increased persistent (late) current, but did not change I(Na) decay. These CaMKII-induced changes of Na+ channel gating were completely abolished by a specific CaMKII inhibitor, autocamtide-2-related inhibitory peptide (AIP). Ca2+/CaM alone reproduced the CaMKII-induced changes of I(Na) availability and the fraction of channels undergoing slow inactivation, but did not alter recovery from inactivation or the magnitude of the late current. Furthermore, the CaM-induced changes were also completely abolished by AIP. On the other hand, cAMP-dependent protein kinase A inhibitors did not abolish the CaM/CaMKII-induced alterations of I(Na) function. CONCLUSION: Ca2+/CaM and CaMKII have distinct effects on the inactivation phenotype of cardiac Na+ channels. The differences are consistent with CaM-independent effects of CaMKII on cardiac Na+ channel gating.

Pyrene-terminated phenylenethynylene rigid linkers anchored to metal oxide nanoparticles.

Phenylenethynylene (PE) rigid linkers (para and meta) were used to anchor pyrene to the surface of TiO2 (anatase) and ZrO2 nanoparticle thin films through the two COOH groups of an isophthalic acid (Ipa) unit. Four chromophore-linker models were studied in solution and bound. Two are novel meta-pyrene-PE linker systems: dimethyl 5-(3-(1-pyrenylethynyl)phenylethynyl)-isophthalate, carrying one pyrene, and dimethyl 5-(bis-3,5-(1-pyrenylethynyl)phenylethynyl)-isophthalate, carrying two. These were compared with para rigid-rods dimethyl 5-(1-pyrenylethynyl)isophthalate and dimethyl 5-(4-(1-pyrenylethynyl)phenylethynyl)-isophthalate, each carrying one pyrene but varying in length. The length of the PE linkers and the para or meta substitution influence the photophysical properties of the compounds. The extinction coefficient increased, and the long wavelength absorbance of the pyrene chromophore was shifted to the red with increasing conjugation. Compared to unsubstituted pyrene, the pyrene-linker systems were characterized by short fluorescence lifetimes (tau approximately 2 ns in tetrahydrofuran solutions), but quantum yields were close to unity. ZINDO/S CI calculations attribute this effect to a switching in the order of the two lowest-lying singlet states of pyrene. High surface coverages, approximately 10(-8) mol/cm2, and carboxylate binding modes on nanostructured TiO2 films were obtained in all cases. The appearance of a pyrene excimer emission on ZrO2, an insulator, indicates that the pyrene-linker system is closely packed (Py-Py < 4 A) on the surface. The fluorescence emission on TiO2 was completely quenched, consistent with quantitative and rapid electron injection into the semiconductor indicating that the pyrene excimer acts as a sensitizer. Photoelectrochemical studies in regenerative solar cells with I3-/I- as the redox mediator indicated near-quantitative conversion of absorbed photons into an electrical current.