Expression of GIRK (Kir3.1/Kir3.4) channels in mouse fibroblast cells with and without beta1 integrins

G protein-activated K+ channel (GIRK) subunits possess a conserved extracellular integrin-binding motif (RGD) and bind directly to beta1 integrins. We expressed GIRK1/GIRK4 channels labeled with green fluorescent protein in fibroblast cell lines expressing or lacking beta1 integrins. Neither plasma membrane localization nor agonist-evoked GIRK currents were affected by the absence of beta1 integrins or by incubation with externally applied RGD-containing peptide. Mutation of the aspartate (D) of RGD impaired currents, GIRK glycosylation, and membrane localization, but the interaction with beta1 integrins remained intact. Thus, beta1 integrins are not essential for functional GIRK expression; and the GIRK-integrin interactions involve structural elements other than the RGD motif.     

PKC-delta sensitizes Kir3.1/3.2 channels to changes in membrane phospholipid levels after M3 receptor activation in HEK-293 cells

G protein-gated inward rectifier (Kir3) channels are inhibited by activation of G_q/11-coupled receptors and this has been postulated to involve the signaling molecules protein kinase C (PKC) and/or phosphatidylinositol 4,5-bisphosphate (PIP₂). Their precise roles in mediating the inhibition of this family of channels remain controversial. We examine here their relative roles in causing inhibition of Kir3.1/3.2 channels stably expressed in human embryonic kidney (HEK)-293 cells after muscarinic M₃ receptor activation. In perforated patch mode, staurosporine prevented the G_q/11-mediated, M₃ receptor, inhibition of channel activity. Recovery from M₃-mediated inhibition was wortmannin sensitive. Whole cell currents, where the patch pipette was supplemented with PIP₂, were still irreversibly inhibited by M₃ receptor stimulation. When adenosine A₁ receptors were co-expressed, inclusion of PIP₂ rescued the A₁-mediated response. Recordings from inside-out patches showed that catalytically active PKC applied directly to the intracellular membrane face inhibited the channels: a reversible effect modulated by okadaic acid. Generation of mutant heteromeric channel Kir3.1S185A/Kir3.2C-S178A, still left the channel susceptible to receptor, pharmacological, and direct kinase-mediated inhibition. Biochemically, labeled phosphate is incorporated into the channel. We suggest that PKC- mediates channel inhibition because recombinant PKC- inhibited channel activity, M₃-mediated inhibition of the channel, was counteracted by overexpression of two types of dominant negative PKC- constructs, and, by using confocal microscopy, we have demonstrated translocation of green fluorescent protein-tagged PKC- to the plasma membrane on M₃ receptor stimulation. Thus Kir3.1/3.2 channels are sensitive to changes in membrane phospholipid levels but this is contingent on the activity of PKC- after M₃ receptor activation in HEK-293 cells. phosphatidylinositol 4,5-bisphosphate; phorbol 12-myristate 13-acetate; receptor for activated C kinase; A kinase anchoring protein; carbachol; 5'-N-ethylcarboxyamidoadenosine     

Expression, localization and functions in acrosome reaction and sperm motility of Ca(V)3.1 and Ca(V)3.2 channels in sperm cells: an evaluation from Ca(V)3.1 and Ca(V)3.2 deficient mice

In spermatozoa, voltage-dependent calcium channels (VDCC) have been involved in different cellular functions like acrosome reaction (AR) and sperm motility. Multiple types of VDCC are present and their relative contribution is still a matter of debate. Based mostly on pharmacological studies, low-voltage-activated calcium channels (LVA-CC), responsible of the inward current in spermatocytes, were described as essential for AR in sperm. The development of Ca(V)3.1 or Ca(V)3.2 null mice provided the opportunity to evaluate the involvement of such LVA-CC in AR and sperm motility, independently of pharmacological tools. The inward current was fully abolished in spermatogenic cells from Ca(V)3.2 deficient mice. This current is thus only due to Ca(V)3.2 channels. We showed that Ca(V)3.2 channels were maintained in sperm by Western-blot and immunohistochemistry experiments. Calcium imaging experiments revealed that calcium influx in response to KCl was reduced in Ca(V)3.2 null sperm in comparison to control cells, demonstrating that Ca(V)3.2 channels were functional. On the other hand, no difference was noticed in calcium signaling induced by zona pellucida. Moreover, neither biochemical nor functional experiments, suggested the presence of Ca(V)3.1 channels in sperm. Despite the Ca(V)3.2 channels contribution in KCl-induced calcium influx, the reproduction parameters remained intact in Ca(V)3.2 deficient mice. These data demonstrate that in sperm, besides Ca(V)3.2 channels, other types of VDCC are activated during the voltage-dependent calcium influx of AR, these channels likely belonging to high-voltage activated Ca(2+) channels family. The conclusion is that voltage-dependent calcium influx during AR is due to the opening of redundant families of calcium channels.     

A switch mechanism for G beta gamma activation of I(KACh)

G protein-gated inwardly rectifying potassium (GIRK) channels are a family of K(+)-selective ion channels that slow the firing rate of neurons and cardiac myocytes. GIRK channels are directly bound and activated by the G protein G beta gamma subunit. As heterotetramers, they comprise the GIRK1 and the GIRK2, -3, or -4 subunits. Here we show that GIRK1 but not the GIRK4 subunit is phosphorylated when heterologously expressed. We found also that phosphatase PP2A dephosphorylation of a protein in the excised patch abrogates channel activation by G beta gamma. Experiments with the truncated molecule demonstrated that the GIRK1 C-terminal is critical for both channel phosphorylation and channel regulation by protein phosphorylation, but the critical phosphorylation sites were not located on the C terminus. These data provide evidence for a novel switch mechanism in which protein phosphorylation enables G beta gamma gating of the channel complex.     

Aldosterone modulates I(f) current through gene expression in cultured neonatal rat ventricular myocytes

Mineralocorticoid receptor (MR) antagonists decrease the incidence of sudden cardiac death in patients with heart failure, as has been reported in two clinical trials (Randomized Aldactone Evaluation Study and Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study). Aldosterone has been shown to increase the propensity to arrhythmias by changing the expression or function of various ion channels. In this study, we investigate the effect of aldosterone on the expression of hyperpolarization-activated current (I(f)) channels in cultured neonatal rat ventricular myocytes, using the whole cell patch-clamp technique, real-time PCR, and Western blotting. Incubation with 10 nM aldosterone for 17-24 h significantly accelerates the rate of spontaneous beating by increasing diastolic depolarization. I(f) current elicited by hyperpolarization from -50 to -130 mV significantly increases aldosterone by 10 nM (by 1.9-fold). Exposure to aldosterone for 1.5 h increases hyperpolarization-activated cyclic nucleotide-gated (HCN) 2 mRNA by 26.3% and HCN4 mRNA by 47.2%, whereas HCN1 mRNA expression remains unaffected. Aldosterone (24-h incubation) increases the expression of HCN2 protein (by 60.0%) and HCN4 protein (by 84.8%), but not HCN1 protein. MR antagonists (1 microM eplerenone or 0.1 microM spironolactone) abolish the increase of I(f) channel expression (currents, mRNA, and protein levels) by 10 nM aldosterone. In contrast, 1 microM aldosterone downregulated I(f) channel gene expression. Glucocorticoid receptor antagonist (100 nM RU-38486) did not affect the increase of I(f) current by 10 nM aldosterone. These findings suggest that aldosterone in physiological concentrations upregulates I(f) channel gene expression by MR activation in cardiac myocytes and may increase excitability, which may have a potential proarrhythmic bearing under pathophysiological conditions.     

Myristoylated peptides potentiate the funny current (I(f)) in sinoatrial myocytes

The funny current, I(f), in sinoatrial myocytes is thought to contribute to the sympathetic fight-or-flight increase in heart rate. I(f) is produced by hyperpolarization-activated cyclic nucleotide sensitive-4 (HCN4) channels, and it is widely believed that sympathetic regulation of I(f) occurs via direct binding of cAMP to HCN4, independent of phosphorylation. However, we have recently shown that Protein Kinase A (PKA) activity is required for sympathetic regulation of I(f) and that PKA can directly phosphorylate HCN4. In the present study, we examined the effects of a myristoylated PKA inhibitory peptide (myr-PKI) on I(f) in mouse sinoatrial myocytes. We found that myr-PKI and another myristoylated peptide potently and specifically potentiated I(f) via a mechanism that did not involve PKA inhibition and that was independent of the peptide sequence, Protein Kinase C, or phosphatidylinositol-4,5-bisphosphate. The off-target activation of I(f) by myristoylated peptides limits their usefulness for studies of pacemaker mechanisms in sinoatrial myocytes.     

The selectivity filter may act as the agonist-activated gate in the G protein-activated Kir3.1/Kir3.4 K+ channel

The Kir3.1/Kir3.4 channel is activated by Gbetagamma subunits released on binding of acetylcholine to the M2 muscarinic receptor. A mechanism of channel opening, similar to that for the KcsA and Shaker K+ channels, has been suggested that involves translocation of pore lining transmembrane helices and the opening of an intracellular gate at the "bundle crossing" region. However, in the present study, we show that an extracellular gate at the selectivity filter is critical for agonist activation of the Kir3.1/Kir3.4 channel. Increasing the flexibility of the selectivity filter, by disrupting a salt bridge that lies directly behind the filter, abolished both selectivity for K+ and agonist activation of the channel. Other mutations within the filter that altered selectivity also altered agonist activation. In contrast, mutations within the filter that did not affect selectivity had little if any effect on agonist activation. Interestingly, mutation of bulky side chain phenylalanine residues at the bundle crossing also altered both agonist activation and selectivity. These results demonstrate a significant correlation between agonist activation and selectivity, which is determined by the selectivity filter, and suggests, therefore, that the selectivity filter may act as the agonist-activated gate in the Kir3.1/Kir3.4 channel.     

The molecular mechanism by which PIP(2) opens the intracellular G-loop gate of a Kir3.1 channel

Inwardly rectifying potassium (Kir) channels are characterized by a long pore comprised of continuous transmembrane and cytosolic portions. A high-resolution structure of a Kir3.1 chimera revealed the presence of the cytosolic (G-loop) gate captured in the closed or open conformations. Here, we conducted molecular-dynamics simulations of these two channel states in the presence and absence of phosphatidylinositol bisphosphate (PIP(2)), a phospholipid that is known to gate Kir channels. Simulations of the closed state with PIP(2) revealed an intermediate state between the closed and open conformations involving direct transient interactions with PIP(2), as well as a network of transitional inter- and intrasubunit interactions. Key elements in the G-loop gating transition involved a PIP(2)-driven movement of the N-terminus and C-linker that removed constraining intermolecular interactions and led to CD-loop stabilization of the G-loop gate in the open state. To our knowledge, this is the first dynamic molecular view of PIP(2)-induced channel gating that is consistent with existing experimental data.     

Modeling transmural heterogeneity of K(ATP) current in rabbit ventricular myocytes

To investigate the mechanisms regulating excitation-metabolic coupling in rabbit epicardial, midmyocardial, and endocardial ventricular myocytes we extended the LabHEART model (Puglisi JL and Bers DM. Am J Physiol Cell Physiol 281: C2049–C2060, 2001). We incorporated equations for Ca²⁺ and Mg²⁺ buffering by ATP and ADP, equations for nucleotide regulation of ATP-sensitive K⁺ channel and L-type Ca²⁺ channel, Na⁺-K⁺-ATPase, and sarcolemmal and sarcoplasmic Ca²⁺-ATPases, and equations describing the basic pathways (creatine and adenylate kinase reactions) known to communicate the flux changes generated by intracellular ATPases. Under normal conditions and during 20 min of ischemia, the three regions were characterized by different I_Na, I_to, I_Kr, I_Ks, and I_Kp channel properties. The results indicate that the ATP-sensitive K⁺ channel is activated by the smallest reduction in ATP in epicardial cells and largest in endocardial cells when cytosolic ADP, AMP, PCr, Cr, P_i, total Mg²⁺, Na⁺, K⁺, Ca²⁺, and pH diastolic levels are normal. The model predicts that only K_ATP ionophore (Kir6.2 subunit) and not the regulatory subunit (SUR2A) might differ from endocardium to epicardium. The analysis suggests that during ischemia, the inhomogeneous accumulation of the metabolites in the tissue sublayers may alter in a very irregular manner the K_ATP channel opening through metabolic interactions with the endogenous PI cascade (PIP₂, PIP) that in turn may cause differential action potential shortening among the ventricular myocyte subtypes. The model predictions are in qualitative agreement with experimental data measured under normal and ischemic conditions in rabbit ventricular myocytes. ATP-sensitive K⁺ channel; creatine and adenylate kinase reactions; phosphatidylinositol phosphates; heart; mathematical model     

Role of I(K) and I(f) in the pacemaker mechanisms of sino-atrial node myocytes

The role of I(K) (delayed rectifier current) and I(f) (hyperpolarization-activated current) in dominant and subsidiary pacemaker ranges was studied in single myocytes isolated from the guinea pig sino-atrial node by means of a perforated patch-clamp technique. In the dominant pacemaker range (approx. -55 to -40 mV), I(K) tails are present whereas I(f) is not activated. In the subsidiary pacemaker range (approx. -80 to -70 mV), I(f) is large whereas I(K) is minimal and reversing. The threshold for I(f) activation is more negative at short time intervals. Larger or longer depolarizations to -40 mV and +20 mV deactivate I(f) more and are followed by faster reactivation of I(f). Steps of 200-300 ms duration to +20 mV completely deactivate I(f). The slope conductance decreases during depolarizations at -40 and +20 mV and quickly re-increases after the steps. The I(f) deactivation range is between -70 and +10 mV, with a V(1/2) of -35 mV. Depolarizations from -80 to +20 mV at a rate of 120/min limit the subsequent I(f) reactivation owing to the short diastole. We conclude that I(K) plays a predominant role in the dominant pacemaker range and I(f) does so in the subsidiary pacemaker range. Either pacemaker mechanism is used by sino-atrial node cells depending on the diastolic potential range. A previous depolarization markedly increases the amplitude and rate of I(f) reactivation.     

Distribution and characterization of cyclo(His-Pro)-like immunoreactivity in the rat gastrointestinal tract

The distribution of cyclo(His-Pro), thyrotropin-releasing hormone (TRH) and $\text{pyroglutamate aminopeptidase}$ activity was examined in the rat gastrointestinal (GI) tract. Cyclo(His-Pro)-like immunoreactivity was present in the following order of distribution (fmoles/mg protein): caecum > colon = jejunum = ileum > stomach = duodenum = rectum, and was immunologically and chromatographically identical with the authentic cyclo(His-Pro). Cyclo(His-Pro) concentrations showed significantly positive correlations with TRH concentrations, but not with $\text{pyroglutamate aminopeptidase}$ activities, in most tissues of the GI tract, suggesting a precursor role of TRH for gut cyclo(His-Pro). These data suggest that cyclo(His-Pro) may be involved in regulating rat GI functions.     

Pituitary adenylate cyclase-activating polypeptide activates K(ATP) current in rat atrial myocytes

Because the electrophysiological effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on the heart are little known, we studied the regulation of the atrial ATP-sensitive K(+) (K(ATP)) current by PACAP on primary cultured neonatal rat atrial myocytes. PACAP-38 stimulates cAMP production with EC(50) = 0.28 nmol/l (r = 0.92, P < 0.02). PACAP-38 and PACAP-27 (10 nmol/l) have similar maximal effects, whereas 100 nmol/l vasoactive intestinal polypeptide (VIP) is 2.7 times less effective (P < 0.05). RT-PCR shows the presence of cloned PACAP receptors PAC(1) (> or =2 isoforms), VPAC(1), and VPAC(2). PACAP-38 dose dependently activates the whole cell atrial K(ATP) current with EC(50) = 1-3 nmol/l (n = 44). Maximal effects occur at 10 nmol/l (91 +/- 15 pA/pF, n = 18). Diazoxide further increases the PACAP-activated current by 78% (P < 0.05; n = 6). H(89) (500 nmol/l), a protein kinase A (PKA) inhibitor, reduces the PACAP-activated K(ATP) current to 17.8 +/- 9.6% (n = 5) of the maximal diazoxide-induced current and totally inhibits the cAMP-induced K(ATP) current. A protein kinase C (PKC) inhibitor peptide (50 micromol/l) in the pipette reduces the PACAP-38-induced K(ATP) current to 33 +/- 17 pA/pF (P < 0.05, n = 6) without significantly affecting the currents induced by cAMP or VIP. The results suggest that: 1) PAC(1), VPAC(1), and VPAC(2) are present in atrial myocytes; and 2) PACAP-38 activates the atrial K(ATP) channels through both PKA and PKC pathways.     

Characterization of an lrp -like ( IrpC ) gene from Bacillus subtilis

In the course of the Bacillus subtilis genome sequencing project, we identified an open reading frame encoding a putative 16.4 kDa protein. This protein shows, respectively, 34% and 25% identity with the Escherichia coli regulatory proteins Lrp and AsnC. Phylogenetic analysis suggests that it represents a new group in the AsnC-Lrp family. Sequence comparisons, as well as immunodetection experiments, lead to the conclusion that the product of this B. subtilislrp-likegene is a bona fide Lrp protein – the first one to be detected in gram-positive bacteria. When expressed in E.␣coli, the B. subtilis Lrp-like protein is able to repress, by about two-fold, the expression of the ilvIH operon which is normally regulated by E. coli Lrp, indicating functional similarity in their regulatory targets. Vegetative growth of a B. subtilis lrp-like mutant is not affected in rich medium. However, the lrp-like mutation causes a transitory inhibition of growth in minimal medium in the presence of valine and isoleucine, which is relieved by leucine. This points to a possible role in regulation of amino acid metabolism. In addition, sporogenesis occurs earlier in the lrp-like mutant than in the reference strain, implying that the B. subtilis Lrp-like protein plays a role in the growth phase transition. Received: 28 January 1997 / Accepted: 18 April 1997     

Putative ACP phosphodiesterase gene (acpD) encodes an azoreductase

An FMN-dependent NADH-azoreductase of Escherichia coli was purified and analyzed for identification of the gene responsible for azo reduction by microorganisms. The N-terminal sequence of the azoreductase conformed to that of the acpD gene product, acyl carrier protein phosphodiesterase. Overexpression of the acpD gene provided the E. coli with a large amount of the 23-kDa protein and more than 800 times higher azoreductase activity. The purified gene product exhibited activity corresponding to that of the native azoreductase. The reaction followed a ping-pong mechanism requiring 2 mol of NADH to reduce 1 mol of methyl red (4'-dimethylaminoazobenzene-2-carboxylic acid) into 2-aminobenzoic acid and N,N'-dimethyl-p-phenylenediamine. On the other hand, the gene product could not convert holo-acyl carrier protein into the apo form under either in vitro or in vivo conditions. These data indicate that the acpD gene product is not acyl carrier protein phosphodiesterase but an azoreductase.