Search

Gap junctions confer interconnectivity of the cytoplasm in neighboring cells via docking of two connexons expressed in each of the adjacent membranes. Undocked connexons, referred to as hemichannels, may open and connect the cytoplasm with the extracellular fluid. The hemichannel configuration of connexins (Cxs) displays isoform-specific permeability profiles that are not directly determined by the size and charge of the permeant. To further explore Ca2+-mediated gating and permeability features of connexin hemichannels, we heterologously expressed Cx30 hemichannels in Xenopus laevis oocytes. The sensitivity toward divalent cation-mediated gating differed between small atomic ions (current) and fluorescent dye permeants, indicating that these permeants are distinctly gated. Three aspartate residues in Cx30 (Asp-50, Asp-172, and Asp-179) have been implicated previously in the Ca2+ sensitivity of other hemichannel isoforms. Although the aspartate at position Asp-50 was indispensable for divalent cation-dependent gating of Cx30 hemichannels, substitutions of the two other residues had no significant effect on gating, illustrating differences in the gating mechanisms between connexin isoforms. Using the substituted cysteine accessibility method (SCAM), we evaluated the role of possible pore-lining residues in the permeation of ions and ethidium through Cx30 hemichannels. Of the cysteine-substituted residues, interaction of a proposed pore-lining cysteine at position 37 with the positively charged compound [2-(trimethylammonium)ethyl] methane thiosulfonate bromide (MTS-ET) increased Cx30-mediated currents with unperturbed ethidium permeability. In summary, our results demonstrate that the permeability of hemichannels is regulated in a permeant-specific manner and underscores that hemichannels are selective rather than non-discriminating and freely diffusable pores.

The function of the KCNE5 (KCNE1-like) protein has not previously been described. Here we show that KCNE5 induces both a time- and voltage-dependent modulation of the KCNQ1 current. Interaction of the KCNQ1 channel with KCNE5 shifted the voltage activation curve of KCNQ1 by more than 140 mV in the positive direction. The activation threshold of the KCNQ1+KCNE5 complex was +40 mV and the midpoint of activation was +116 mV. The KCNQ1+KCNE5 current activated slowly and deactivated rapidly as compared to the KCNQ1+KCNE1 at 22 degrees C; however, at physiological temperature, the activation time constant of the KCNQ1+KCNE5 current decreased fivefold, thus exceeding the activation rate of the KCNQ1+KCNE1 current. The KCNE5 subunit is specific for the KCNQ1 channel, as none of other members of the KCNQ-family or the human ether a-go-go related channel (hERG1) was affected by KCNE5. Four residues in the transmembrane domain of the KCNE5 protein were found to be important for the control of the voltage-dependent activation of the KCNQ1 current. We speculate that since KCNE5 is expressed in cardiac tissue it may here along with the KCNE1 beta-subunit regulate KCNQ1 channels. It is possible that KCNE5 shapes the I(Ks) current in certain parts of the mammalian heart.

Background: Diabetes and metabolic syndrome causes cardiac electrical abnormalities that increase the risk for arrhythmias and sudden death. We have established fructose-fat fed rats (FFFRs) as a prediabetic model, which after 6 weeks of feeding exhibit intramyocardial lipid accumulation, decreased cardiac conduction velocity (CV), and increased risk of ventricular fibrillation following ischemia–reperfusion. CV slowing was not explained by changes in Naþ, Kþ, or gap junction channel function; however, CV may be altered by a depolarized resting membrane potential (RMP). Therefore, the aim of this study was to investigate the time dependence of diet-induced cardiac lipid accumulation, CV disturbances, and changes in RMP and action potential (AP) morphology. Methods: Four-week-old male Sprague-Dawley rats were fed ad libitum with either normal chow or a diet consisting of 60% fat in the chow and 10% fructose in the drinking water. After 1, 3, and 6 weeks of feeding, we measured cardiac CV in right ventricular tissue strips with extracellular electrodes, AP morphology, and RMP using intracellular microelectrodes, and triglyceride content by thin layer chromatography. Results: CV increased from 68.5 0.24 cm/s to 77.7 0.23 cm/s from 1 to 6 weeks in control rats, whereas FFFRs failed to increase CV during the same period (68.9 0.23 cm/s after 1 week vs 67.9 0.23 cm/s after 6 weeks). Intramyocardial triglyceride levels were already significantly higher in FFFRs compared to controls, after 3 weeks of feeding, a time point when CV was not significantly different between controls and FFFRs (70.5 0.15 cm/s vs 70.3 0.20 cm/s). RMP and AP morphology were not different between the 2 groups at any point, except for time to 20% repolarization (APD20), which was significantly longer for FFFRs after 6 weeks (5.69 0.33 ms vs 4.62 0.43 ms). Conclusions: Rats on normal chow increase CV between 1 and 6 weeks of feeding (approximate age 5–6 and 10–11 weeks, respectively), whereas fructose-fat feeding prevents CV development. Lipid accumulation is already present after 3 weeks of fructose-fat feeding, preceding CV slowing. CV slowing could not be explained by changes in RMP or AP upstroke velocity.