Background: SecA has been viewed as ATPase helping precursors across SecYEG channels. Results: SecA alone could promote protein translocation and ion channel activity, but loses specificity and efficiency, which can be restored by SecYEG. Conclusion: SecA plays important structural roles and can function as low affinity protein conducting channels in membranes. Significance: Establishing SecA as channels is crucial for understanding diverse mechanisms and evolution of bacterial translocation pathways.

CO2 chemoreception may be mediated by the modulation of certain ion channels in neurons. Kir4.1 and Kir5.1, two members of the inward rectifier K+ channel family, are expressed in several brain regions including the brainstem. To test the hypothesis that Kir4.1 and Kir5.1 are modulated by CO2 and pH, we carried out experiments by expressing Kir4.1 and coexpressing Kir4.1 with Kir5.1 (Kir4.1‐Kir5.1) in Xenopus oocytes. K+ currents were then studied using two‐electrode voltage clamp and excised patches. Exposure of the oocytes to CO2 (5, 10 and 15 %) produced a concentration‐dependent inhibition of the whole‐cell K+ currents. This inhibition was fast and reversible. Exposure to 15 % CO2 suppressed Kir4.1 currents by ∼20 % and Kir4.1‐Kir5.1 currents by ∼60 %. The effect of CO2 was likely to be mediated by intracellular acidification, because selective intracellular, but not extracellular, acidification to the measured hypercapnic pH levels lowered the currents as effectively as hypercapnia. In excised inside‐out patches, exposure of the cytosolic side of membranes to solutions with various pH levels brought about a dose‐dependent inhibition of the macroscopic K+ currents. The pK value (‐log of dissociation constant) for the inhibition was 6.03 in the Kir4.1 channels, while it was 7.45 in Kir4.1‐Kir5.1 channels, an increase in pH sensitivity of 1.4 pH units. Hypercapnia without changing pH did not inhibit the Kir4.1 and Kir4.1‐Kir5.1 currents, suggesting that these channels are inhibited by protons rather than molecular CO2. A lysine residue in the N terminus of Kir4.1 is critical. Mutation of this lysine at position 67 to methionine (K67M) completely eliminated the CO2 sensitivity of both the homomeric Kir4.1 and heteromeric Kir4.1‐Kir5.1. These results therefore indicate that the Kir4.1 channel is inhibited during hypercapnia by a decrease in intracellular pH, and the coexpression of Kir4.1 with Kir5.1 greatly enhances channel sensitivity to CO2/pH and may enable cells to detect both increases and decreases in PCO2 and intracellular pH at physiological levels.

channels have a higher K¤ permeability at hyperpolarizing than depolarizing membrane potentials. Activity of these K¤ channels is also controlled by several intra-and extracellular factors. One of these factors is the hydrogen ion (Coulter et al. 1995;Tsai et al. 1995;Doi et al. 1996;Fakler et al. 1996;Shieh et al. 1996;Choe et al. 1997;Sabirov et al. 1997). While H¤ concentration, or pH level, is maintained by intra-and extracellular buffer systems, there are conditions in which protons are overproduced during respiratory or metabolic acidosis. A decrease in intra-or extracellular pH has been shown to inhibit inward rectifier K¤ channels in

People with Rett syndrome (RTT) have breathing instability in addition to other neuropathological manifestations. The breathing disturbances contribute to the high incidence of unexplained death and abnormal brain development. However, the cellular mechanisms underlying the breathing abnormalities remain unclear. To test the hypothesis that the central CO(2) chemoreception in these people is disrupted, we studied the CO(2) chemosensitivity in a mouse model of RTT. The Mecp2-null mice showed a selective loss of their respiratory response to 1-3% CO(2) (mild hypercapnia), whereas they displayed more regular breathing in response to 6-9% CO(2) (severe hypercapnia). The defect was alleviated with the NE uptake blocker desipramine (10 mg·kg(-1)·day(-1) ip, for 5-7 days). Consistent with the in vivo observations, in vitro studies in brain slices indicated that CO(2) chemosensitivity of locus coeruleus (LC) neurons was impaired in Mecp2-null mice. Two major neuronal pH-sensitive Kir currents that resembled homomeric Kir4.1 and heteromeric Ki4.1/Kir5.1 channels were identified in the LC neurons. The screening of Kir channels with real-time PCR indicated the overexpression of Kir4.1 in the LC region of Mecp2-null mice. In a heterologous expression system, an overexpression of Kir4.1 resulted in a reduction in the pH sensitivity of the heteromeric Kir4.1-Kir5.1 channels. Given that Kir4.1 and Kir5.1 subunits are also expressed in brain stem respiration-related areas, the Kir4.1 overexpression may not allow CO(2) to be detected until hypercapnia becomes severe, leading to periodical hyper- and hypoventilation in Mecp2-null mice and, perhaps, in people with RTT as well.

CO2 chemoreception may be related to modulation of inward rectifier K+ channels (Kir channels) in brainstem neurons. Kir4.1 is expressed predominantly in the brainstem and inhibited during hypercapnia. Although the homomeric Kir4.1 only responds to severe intracellular acidification, coexpression of Kir4.1 with Kir5.1 greatly enhances channel sensitivities to CO2 and pH. To understand the biophysical and molecular mechanisms underlying the modulation of these currents by CO2 and pH, heteromeric Kir4.1–Kir5.1 were studied in inside-out patches. These Kir4.1–Kir5.1 currents showed a single channel conductance of 59 pS with open-state probability (P open) ∼ 0.4 at pH 7.4. Channel activity reached the maximum at pH 8.5 and was completely suppressed at pH 6.5 with pKa 7.45. The effect of low pH on these currents was due to selective suppression of P open without evident effects on single channel conductance, leading to a decrease in the channel mean open time and an increase in the mean closed time. At pH 8.5, single-channel currents showed two sublevels of conductance at ∼1/4 and 3/4 of the maximal openings. None of them was affected by lowering pH. The Kir4.1–Kir5.1 currents were modulated by phosphatidylinositol-4,5-bisphosphate (PIP2) that enhanced baseline P open and reduced channel sensitivity to intracellular protons. In the presence of 10 μM PIP2, the Kir4.1–Kir5.1 showed a pKa value of 7.22. The effect of PIP2, however, was not seen in homomeric Kir4.1 currents. The CO2/pH sensitivities were related to a lysine residue in the NH2 terminus of Kir4.1. Mutation of this residue (K67M, K67Q) completely eliminated the CO2 sensitivity of both homomeric Kir4.1 and heteromeric Kir4.1–Kir5.1. In excised patches, interestingly, the Kir4.1–Kir5.1 carrying K67M mutation remained sensitive to low pHi. Such pH sensitivity, however, disappeared in the presence of PIP2. The effect of PIP2 on shifting the titration curve of wild-type and mutant channels was totally abolished when Arg178 in Kir5.1 was mutated. Thus, these studies demonstrate a heteromeric Kir channel that can be modulated by both acidic and alkaline pH, show the modulation of pH sensitivity of Kir channels by PIP2, and provide information of the biophysical and molecular mechanisms underlying the Kir modulation by intracellular protons.

ATP-sensitive K(+) (K(ATP)) channels are activated by several vasodilating hormones and neurotransmitters through the PKA pathway. Here, we show that phosphorylation at Ser1387 of the SUR2B subunit is critical for the channel activation. Experiments were performed in human embryonic kidney (HEK) 293 cells expressing the cloned Kir6.1/SUR2B channel. In whole cell patch, the Kir6.1/SUR2B channel activity was stimulated by isoproterenol via activation of beta(2) receptors. This effect was blocked in the presence of inhibitors for adenylyl cyclase or PKA. Similar channel activation was seen by exposing inside-out patches to the catalytic subunit of PKA. Because none of the previously suggested PKA phosphorylation sites accounted for the channel activation, we performed systematic mutational analysis on Kir6.1 and SUR2B. Two serine residues (Ser1351, Ser1387) located in the NBD2 of SUR2B were critical for the channel activation. In vitro phosphorylation experiments showed that Ser1387 but not Ser1351 was phosphorylated by PKA. The PKA-dependent activation of cell-endogenous K(ATP) channels was observed in acutely dissociated mesenteric smooth myocytes and isolated mesenteric artery rings, where activation of these channels contributed significantly to the isoproterenol-induced vasodilation. Taken together, these results indicate that the Kir6.1/SUR2B channel is a target of beta(2) receptors and that the channel activation relies on PKA phosphorylation of SUR2B at Ser1387.

Rett syndrome caused by mutations in methyl-CpG-binding protein 2 (Mecp2) gene shows abnormalities in autonomic functions in which brain stem norepinephrinergic systems play an important role. Here we present systematic comparisons of intrinsic membrane properties of locus coeruleus (LC) neurons between Mecp2(-/Y) and wild-type (WT) mice. Whole cell current clamp was performed in brain slices of 3- to 4-wk-old mice. Mecp2(-/Y) neurons showed stronger inward rectification and had shorter time constant than WT cells. The former was likely due to overexpression of inward rectifier K(+) (K(ir))4.1 channel, and the latter was attributable to the smaller cell surface area. The action potential duration was prolonged in Mecp2(-/Y) cells with an extended rise time. This was associated with a significant reduction in the voltage-activated Na(+) current density. After action potentials, >60% Mecp2(-/Y) neurons displayed fast and medium afterhyperpolarizations (fAHP and mAHP), while nearly 90% WT neurons showed only mAHP. The mAHP amplitude was smaller in Mecp2(-/Y) neurons. The firing frequency was higher in neurons with mAHP, and the frequency variation was greater in cells with both fAHP and mAHP in Mecp2(-/Y) mice. Small but significant differences in spike frequency adaptation and delayed excitation were found in Mecp2(-/Y) neurons. These results indicate that there are several electrophysiological abnormalities in LC neurons of Mecp2(-/Y) mice, which may contribute to the dysfunction of the norepinephrine system in Rett syndrome.

ATP-sensitive K؉ (K ATP ) channels may be regulated by protons in addition to ATP, phospholipids, and other nucleotides. Such regulation allows a control of cellular excitability in conditions when pH is low but ATP concentration is normal. However, whether the K ATP changes its activity with pH alterations remains uncertain. In this study we showed that the reconstituted K ATP was strongly activated during hypercapnia and intracellular acidosis using whole-cell recordings. Further characterizations in excised patches indicated that channel activity increased with a moderate drop in intracellular pH and decreased with strong acidification. The channel activation was produced by a direct action of protons on the Kir6 subunit and relied on a histidine residue that is conserved in all K ATP . The inhibition appeared to be a result of channel rundown and was not seen in whole-cell recordings. The biphasic response may explain the contradictory pH sensitivity observed in cell-endogenous K ATP in excised patches. Site-specific mutations of two residues showed that pH and ATP sensitivities were independent of each other. Thus, these results demonstrate that the proton is a potent activator of the K ATP . The pH-dependent activation may enable the K ATP to control vascular tones, insulin secretion, and neuronal excitability in several pathophysiologic conditions. Hypercapnia and acidosis affect vascular tone, skeletal muscle contractility, insulin secretion, epithelial transport, and neuronal excitability, which may be mediated by K ATP 1 (1-5). However, previous studies on the pH sensitivity of these K ϩ channels were controversial and even contradictory. In the absence of ATP, acidic pH was shown to stimulate cell-endogenous K ATP (6, 7), inhibit it (8, 9), and have little or no effect (10, 11). This inconsistency is further complicated by the indirect effect of ATP or Mg 2ϩ and tissue-specific K ATP species (8 -12). Consequently, it is unclear whether K ATP is modulated during hypercapnia and acidosis and what molecular mechanisms are underlying the modulations. The cloned K ATP channels are ideal for addressing these questions because they allow for fine dissection of the modulatory mechanisms and elaborate manipulation of PCO 2 and pH in an expression system (13,14). Therefore, we studied the modulation of the cloned K ATP (Kir6 with SUR, Ref. 15) by CO 2 and acidic pH. To locate the pH sensors, we also studied Kir6.2 with a truncation of 36 amino acids at the C terminus (Kir6.2⌬C36) because it expresses functional channel without the SUR subunit and retains fair ATP sensitivity (16). MATERIALS AND METHODSOocytes from Xenopus laevis were used in the present studies. Frogs were anesthetized by bathing them in 0.3% 3-aminobenzoic acid ethyl ester. A few lobes of ovaries were removed after a small abdominal incision (ϳ5 mm). Then, the surgical incision was closed and the frogs were allowed to recover from the anesthesia. Xenopus oocytes were treated with 2 mg/ml collagenase (Type I, Sigma) in an OR2 solution consistin...