Modulation of calcium-sensitive potassium (BK) channels by oxygen is important in several mammalian tissues, and in the carotid body it is crucial to respiratory control. However, the identity of the oxygen sensor remains unknown. We demonstrate that hemoxygenase-2 (HO-2) is part of the BK channel complex and enhances channel activity in normoxia. Knockdown of HO-2 expression reduced channel activity, and carbon monoxide, a product of HO-2 activity, rescued this loss of function. Inhibition of BK channels by hypoxia was dependent on HO-2 expression and was augmented by HO-2 stimulation. Furthermore, carotid body cells demonstrated HO-2-dependent hypoxic BK channel inhibition, which indicates that HO-2 is an oxygen sensor that controls channel activity during oxygen deprivation.

NO-induced activation of cGMP-dependent protein kinase (PKG) increases the open probability of large conductance Ca2؉ -activated K ؉ channels and results in smooth muscle relaxation. However, the molecular mechanism of channel regulation by the NO-PKG pathway has not been determined on cloned channels. The present study was designed to clarify PKG-mediated modulation of channels at the molecular level. The cDNA encoding the ␣-subunit of the large conductance Large conductance Ca 2ϩ -activated K ϩ (BK Ca ) 1 channels are ubiquitously distributed among tissues and are particularly abundant in smooth muscle (1, 2). The activity of BK Ca channels is regulated by membrane potential, intracellular Ca 2ϩ , and phosphorylation (3, 4). Although BK Ca channels are usually not involved in setting resting potential, they play a key role as a negative feedback mechanism to limit depolarization and contraction (5-7). Activation of BK Ca channels is increased by nitric oxide (NO) and atrial natriuretic peptide, which hyperpolarize the membrane and increase the sensitivity of BK Ca channels to Ca 2ϩ (8 -11). Membrane hyperpolarization closes voltage-dependent Ca 2ϩ channels, reduces Ca 2ϩ influx, and leads to a reduction in intracellular Ca 2ϩ concentration and relaxation (1). NO has been reported to stimulate BK Ca channels directly as well as through stimulation of guanylate cyclase and the subsequent increase in cGMP (12-15). In addition, activation of BK Ca channels plays an important role in NO-induced relaxation of smooth muscle (16 -20). cGMP activates cGMP-dependent protein kinase (PKG), which phosphorylates various cytosolic and membrane proteins that regulate smooth muscle tone either directly or indirectly (21,22). Recent studies in native cells suggest that PKG activates BK Ca channels through phosphorylation of the channel (23). These results are supported by biochemical studies of cloned BK Ca channels, which demonstrate PKG-induced phosphorylation of the channel (24).The primary sequence of BK Ca has been determined using molecular cloning techniques in Drosophila (25) and mammals (26 -28). These studies indicate that BK Ca isoforms belong to the voltage-gated K ϩ (K V ) channel superfamily. The primary sequence of the S1-S6 segment of BK Ca channels is homologous to the corresponding regions in K V channels. The long carboxyl terminus is the region of Ca 2ϩ -sensing (29, 30), and cslo-␣ contains a single high affinity phosphorylation site for PKG at Ser-1072 (3). However additional putative PKG phosphorylation sites have been identified in other splice variants (31). Expression of the slo channel in Xenopus oocytes or mammalian cells gives rise to voltage-gated, Ca 2ϩ -sensitive currents with electrophysiological and pharmacological features similar to those of native BK Ca (32-34). However, although many studies of native cells suggest that BK Ca channel activity is also modulated by various protein kinases (35-38), this property has been difficult to reproduce in cloned channels. Two studies in which slo chann...

Experiments were performed to determine whether capacitative Ca(2+) entry (CCE) can be activated in canine pulmonary and renal arterial smooth muscle cells (ASMCs) and whether activation of CCE parallels the different functional structure of the sarcoplasmic reticulum (SR) in these two cell types. The cytosolic [Ca(2+)] was measured by imaging fura-2-loaded individual cells. Increases in the cytosolic [Ca(2+)] due to store depletion in pulmonary ASMCs required simultaneous depletion of both the inositol 1,4,5-trisphosphate (InsP(3))- and ryanodine (RY)-sensitive SR Ca(2+) stores. In contrast, the cytosolic [Ca(2+)] rises in renal ASMCs occurred when the SR stores were depleted through either the InsP(3) or RY pathways. The increase in the cytosolic [Ca(2+)] due to store depletion in both pulmonary and renal ASMCs was present in cells that were voltage clamped and was abolished when cells were perfused with a Ca(2+)-free bathing solution. Rapid quenching of the fura-2 signal by 100 microM Mn(2+) following SR store depletion indicated that extracellular Ca(2+) entry increased in both cell types and also verified that activation of CCE in pulmonary ASMCs required the simultaneous depletion of the InsP(3)- and RY-sensitive SR Ca(2+) stores, while CCE could be activated in renal ASMCs by the depletion of either of the InsP(3)- or RY-sensitive SR stores. Store depletion Ca(2+) entry in both pulmonary and renal ASMCs was strongly inhibited by Ni(2+) (0.1-10 mM), slightly inhibited by Cd(2+) (200-500 microM), but was not significantly affected by the voltage-gated Ca(2+) channel (VGCC) blocker nisoldipine (10 microM). The non-selective cation channel blocker Gd(3+) (100 microM) inhibited a portion of the Ca(2+) entry in 6 of 18 renal but not pulmonary ASMCs. These results provide evidence that SR Ca(2+) store depletion activates CCE in parallel with the organization of intracellular Ca(2+) stores in canine pulmonary and renal ASMCs.

No abstract

Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites of cytochrome P450 monooxygenase, which are released from endothelial cells and dilate arteries. Dilation seems to be caused by activation of large-conductance Ca2+ activated K+ channels (BK(Ca)) leading to membrane hyperpolarization. Previous studies suggest that EETs activate BK(Ca) channels via ADP-ribosylation of the G protein Galphas with a subsequent membrane-delimited action on the channel [Circ Res 78:415-423, 1996; 80:877-884, 1997; 85:349-356, 1999]. The present study examined whether this pathway is present in human embryonic kidney (HEK) 293 cells when the BK(Ca) alpha-subunit (cslo-alpha) is expressed without the beta-subunit. 11,12-EET increased outward K+ current in whole-cell recordings of HEK293 cells. In cell-attached patches, 11,12-EET also increased the activity of cslo-alpha channels without affecting unitary conductance. This action was mimicked by cholera toxin. The ADP-ribosyltransferase inhibitors 3-aminobenzamide and m-iodobenxylguanidine blocked the stimulatory effect of 11,12-EET. In inside-out patches 11,12-EET was without effect on channel activity unless GTP was included in the bathing solution. GTP and GTPgammaS alone also activated cslo-alpha channels. Dialysis of cells with anti-Galphas antibody completely blocked the activation of cslo-alpha channels by 11,12-EET, whereas anti-Galphai/o and anti-Gbetagamma antibodies were without effect. The protein kinase A inhibitor KT5720 and the adenylate cyclase inhibitor SQ22536 did not reduce the stimulatory effect of 11,12-EET on cslo-alpha channels in cell-attached patches. These data suggest that EET leads to Galphas-dependent activation of the cslo-alpha subunits expressed in HEK293 cells and that the cslo-beta subunit is not required.

Located within the gastrointestinal (GI) musculature are networks of cells known as interstitial cells of Cajal (ICC). ICC are associated with several functions including pacemaker activity that generates electrical slow waves and neurotransmission regulating GI motility. In this study we identified a voltage‐dependent K+ channel (Kv1.1) expressed in ICC and neurons but not in smooth muscle cells. Transcriptional analyses demonstrated that Kv1.1 was expressed in whole tissue but not in isolated smooth muscle cells. Immunohistochemical co‐localization of Kv1.1 with c‐kit (a specific marker for ICC) and vimentin (a specific marker of neurons and ICC) indicated that Kv1.1‐like immunoreactivity (Kv1.1‐LI) was present in ICC and neurons of GI tissues of the dog, guinea‐pig and mouse. Kv1.1‐LI was not observed in smooth muscle cells of the circular and longitudinal muscle layers. Kv1.1 was cloned from a canine colonic cDNA library and expressed in Xenopus oocytes. Pharmacological investigation of the electrophysiological properties of Kv1.1 demonstrated that the mamba snake toxin dendrotoxin‐K (DTX‐K) blocked the Kv1.1 outward current when expressed as a homotetrameric complex (EC50= 0.34 nm). Other Kv channels were insensitive to DTX‐K. When Kv1.1 was expressed as a heterotetrameric complex with Kv1.5, block by DTX‐K dominated, indicating that one or more subunits of Kv1.1 rendered the heterotetrameric channel sensitive to DTX‐K. In patch‐clamp experiments on cultured murine fundus ICC, DTX‐K blocked a component of the delayed rectifier outward current. The remaining, DTX‐insensitive current (i.e. current in the presence of 10−8m DTX‐K) was outwardly rectifying, rapidly activating, non‐inactivating during 500 ms step depolarizations, and could be blocked by both tetraethylammonium (TEA) and 4‐aminopyridine (4‐AP). In conclusion, Kv1.1 is expressed by ICC of several species. DTX‐K is a specific blocker of Kv1.1 and heterotetrameric channels containing Kv1.1. This information is useful as a means of identifying ICC and in studies of the role of delayed rectifier K+ currents in ICC functions.

Two components of voltage‐gated, inward currents were observed from murine colonic myocytes. One component had properties of L‐type Ca2+ currents and was inhibited by nicardipine (5 × 10−7m). A second component did not ‘run down’ during dialysis and was resistant to nicardipine (up to 10−6m). The nicardipine‐insensitive current was activated by small depolarizations above the holding potential and reversed near 0 mV. This low‐voltage‐activated current (ILVA) was resolved with step depolarizations positive to ‐60 mV, and the current rapidly inactivated upon sustained depolarization. The voltage of half‐inactivation was ‐65 mV. Inactivation and activation time constants at ‐45 mV were 86 and 15 ms, respectively. The half‐recovery time from inactivation was 98 ms at ‐45 mV. ILVA peaked at ‐40 mV and the current reversed at 0 mV. I lva was inhibited by Ni2+ (IC50= 1.4 × 10−5m), mibefradil (10−6 to 10−5m), and extracellular Ba2+. Replacement of extracellular Na+ with N‐methyl‐d‐glucamine inhibited ILVA and shifted the reversal potential to ‐7 mV. Increasing extracellular Ca2+ (5 × 10−3m) increased the amplitude of ILVA and shifted the reversal potential to +22 mV. ILVA was also blocked by extracellular Cs+ (10−4m) and Gd3+ (10−6m). Warming increased the rates of activation and deactivation without affecting the amplitude of the peak current. We conclude that the second component of voltage‐dependent inward current in murine colonic myocytes is not a ‘T‐type’ Ca2+ current but rather a novel, voltage‐gated non‐selective cation current. Activation of this current could be important in the recovery of membrane potential following inhibitory junction potentials in gastrointestinal smooth muscle or in mediating responses to agonists.

Human mediator of DNA damage checkpoint 1 (hMDC1) is an essential component of the cellular response to DNA double strand breaks. Recently, hMDC1 has been shown to associate with a subunit of the anaphase-promoting complex/cyclosome (APC/C) (Coster, G., Hayouka, Z., Argaman, L., Strauss, C., Friedler, A., Brandeis, M., and Goldberg, M. (2007) J. Biol. Chem. 282, 32053-32064), a key regulator of mitosis, suggesting a possible role for hMDC1 in controlling normal cell cycle progression. Here, we extend this work to show that hMDC1 regulates normal metaphase-to-anaphase transition through its ability to bind directly to the APC/C and modulate its E3 ubiquitin ligase activity. In support of a role for hMDC1 in controlling mitotic progression, depletion of hMDC1 by small interfering RNA results in a metaphase arrest that appears to be independent of both BubR1-dependent signaling pathways and ATM/ATR activation. Mitotic cells lacking hMDC1 exhibit markedly reduced levels of APC/C activity characterized by reduced levels of Cdc20, and a failure of Cdc20 to bind the APC/C and CREB-binding protein. We suggest therefore that hMDC1 functionally regulates the normal metaphase-to-anaphase transition by modulating the Cdc20-dependent activation of the APC/C. Mitotic progression is regulated by the APC/C, 4 an E3 ubiquitin ligase that controls the ubiquitin-dependent destruction of mitotic cyclins and other substrates in a coordinated manner. The APC/C regulates the metaphase-toanaphase transition principally through promoting Separase activation by mediating the destruction of its inhibitor, Securin, at metaphase (1). APC/C activity and its specificity toward individual substrates are controlled successively during mitosis by the timely binding to one of two closely related activator proteins, Cdc20 and Cdh1. Cdc20-APC/C controls the metaphase-to-anaphase transition, whereas Cdh1-APC/C controls mitotic exit and progression though G 1 (1). More recently, it has been shown that the ability of the APC/C to promote efficient substrate ubiquitylation also requires the presence of CBP, which probably functions through its capacity to act as an E4 ubiquitin ligase (2). To ensure the fidelity of chromosome segregation at anaphase, the activity of Cdc20-APC/C is tightly regulated by proteins that function in the spindle assembly checkpoint, which monitors for the presence of unattached kinetochores. When the spindle checkpoint is activated, Mad2 and BubR1 binding to Cdc20 inhibits Cdc20-APC/C activity and consequently the metaphase-to-anaphase transition (3). The intricacies of mitotic regulation and checkpoint activation are, however, complicated by observations that DNA damage-responsive proteins, such as BRCA1 and Chk1, appear also to function during normal mitosis. Indeed, both BRCA1 and Chk1 reside at centrosomes, with loss of either BRCA1 or Chk1 resulting in premature centrosome separation (4 -6), chromosome misalignment during metaphase, chromosome lagging during anaphase, and kinetochore defects within the regions of misalig...