1. The effects of intracellular pH (pH1) were investigated in inside-out giant patches from Xenopus oocytes expressing the inward rectifier K+ channel IRKI. 2. After excising patches into Mg2+-and polyamine-free solution, a residual time-dependent inactivation of outward current during depolarizing voltage-clamp pulses persisted, reaching an apparent steady-state by 5 min. Raising pH, from 7-2 to 9 0 increased the inactivation rate of the outward current.3. In the presence of intracellular Mg2+ or polyamines, however, pHi 9 0 either decreased or did not change the inactivation rate of outward current. 4. These results suggest that the inactivation of outward current remaining after > 5 min in Mg2+_ and polyamine-free solution is not due to slow washout of these substances, but represents a third and probably intrinsic gating mechanism contributing to the inward rectifying property of IRK1. 6. In the absence of intracellular Mg2+ and polyamines, reduced pHi blocked inward and outward current through IRKI channels in a voltage-independent manner without appreciably altering the kinetics. Half-maximal block occurred at pH 6-2-6-4 (Hill coefficient, 1 6). Block of IRK1 by intracellular protons may contribute to membrane depolarization in ischaemic tissue.

Interactions of Ba2+ with K+ and molecules contributing to inward rectification were studied in the cloned inward rectifier K+ channels, Kir2.1. Extracellular Ba2+ blocked Kir2.1 channels with first-order kinetics in a Vm-dependent manner. At Vm more negative than -120 mV, the Kd-Vm relationship became less steep and the dissociation rate constants were larger, suggesting Ba2+ dissociation into the extracellular space. Both depolarization and increasing [K+]i accelerated the recovery from extracellular Ba2+ blockade. Intracellular K+ appears to relieve Ba2+ blockade by competitively slowing the Ba2+ entrance rate, instead of increasing its exit rate by knocking off action. Intracellular spermine (100 microM) reduced, whereas 1 mM [Mg2+]i only slightly reduced, the ability of intracellular K+ to repulse Ba2+ from the channel pore. Intracellular Ba2+ also blocked outward IKir2.1 in a voltage-dependent fashion. At Vm >/= +40 mV, where intrinsic inactivation is prominent, intracellular Ba2+ accelerated the inactivation rate of the outward IKir2.1 in a Vm-independent manner, suggesting interaction of Ba2+ with the intrinsic gate of Kir2.1 channels.

Heme oxygenase-1 (HO-1), a stress-inducible enzyme anchored in the endoplasmic reticulum (ER) by a single transmembrane segment (TMS) located at the C terminus, interacts with NADPH cytochrome P450 reductase and biliverdin reductase to catalyze heme degradation to biliverdin and its metabolite, bilirubin. Previous studies suggested that HO-1 functions as a monomer. Using chemical cross-linking, co-immunoprecipitation, and fluorescence resonance energy transfer (FRET) experiments, here we showed that HO-1 forms dimers/ oligomers in the ER. However, oligomerization was not observed with a truncated HO-1 lacking the C-terminal TMS (amino acids 266 -285), which exhibited cytosolic and nuclear localization, indicating that the TMS is essential for the selfassembly of HO-1 in the ER. To identify the interface involved in the TMS-TMS interaction, residue Trp-270, predicted by molecular modeling as a potential interfacial residue of TMS ␣-helices, was mutated, and the effects on protein subcellular localization and activity assessed. The results showed that the W270A mutant was present exclusively in the ER and formed oligomers with similar activity to those of the wild type HO-1. Interestingly, the W270N mutant was localized not only in the ER, but also in the cytosol and nucleus, suggesting it is susceptible to proteolytic cleavage. Moreover, the microsomal HO activity of the W270N mutant was significantly lower than that of the wild type. The W270N mutation appears to interfere with the oligomeric state, as revealed by a lower FRET efficiency. Collectively, these data suggest that oligomerization, driven by TMS-TMS interactions, is crucial for the stabilization and function of HO-1 in the ER.Heme oxygenase (HO) 3 catalyzes the NADPH cytochrome P450 reductase-dependent oxidative degradation of cellular heme to biliverdin, carbon monoxide (CO), and free iron (1, 2). Biliverdin is subsequently converted to bilirubin by biliverdin reductase in the cytosol. Two HO isoforms have been identified in mammalian systems. HO-1 is a 288 amino acid protein and is expressed at high amounts in a variety of pathological conditions associated with cellular stress. There is compelling evidence that HO-1 induction represents an important cytoprotective defense mechanism against oxidative insults by virtue of the anti-oxidant properties of the bilirubin and the anti-inflammatory effect of the CO produced (2). HO-1 is anchored in the endoplasmic reticulum (ER) through a single transmembrane segment (TMS) located at the C terminus, while the rest of the molecule is cytoplasmic (3). HO-1 is sensitive to proteolytic cleavage (4), and it was recently shown that HO-1 can be proteolytically cleaved from the ER and translocated to the nucleus under certain stress conditions (5). Although the catalytic site in the cytoplasmic domain remains intact, the activity of soluble HO-1 is drastically reduced (5), indicating that ER localization is important for its full enzymatic function.Self-assembly to form dimers and higher oligomers is a common pheno...

Intracellular permeant anions, and not extracellular protons, are the predominant driver of fast gating in the hyperpolarization-activated CLC-2 chloride channel.

We examined the effects of spermine binding to aspartate at site 172 on the accessibility of internal trimethylammonioethylmethane thiosulphonate (MTSET) to substituted cysteines within the pore of a Kir2.1 channel. Spermine prevented MTSET modification in Q164C and G168C mutants, indicating that sites 164 and 168 are located externally to the spermine binding site. The rates of MTSET modification were significantly reduced by spermine in I176C mutants, indicating that site 176 is located internally to D172 and that the bound spermine hinders the reaction of MTSET with cysteine at site 176. Spermidine, putrescine and Mg2+ also decreased MTSET modification at site 176. The order of effect is putrescine > spermidine ∼ spermine ≈ Mg2+. To account for the electrostatic and physical repulsion between MTSET and polyamines, possible locations of polyamines in the pore are discussed. In D172C mutants, the spermine that bound to sites 224 and 299 completely inhibited channels at +40 mV, yet MTSET remained accessible to site 172. In addition, in the D172C mutant, spermine did not affect the exit rate of Ba2+ bound to the threonine at the site 141. These results indicate that spermine bound at the cytoplasmic pore induces channel closure at positions 141‐172. The effects of spermine on the accessibility of amino acids in the pore may shed light on the structural and functional relationships of the Kir2.1 channels during inward rectification.