PIP₂ modulation of Slick and Slack K⁺ channels

The use of heavy water (D(2)O) as a solvent is commonplace in many spectroscopic techniques for the study of biological macromolecules. A significant deuterium isotope effect exists where hydrogen-bonding is important, such as in protein stability, dynamics and assembly. Here we illustrate the use of D(2)O in additive screening for the production of reproducible diffraction-quality crystals for the Salmonella enteritidis fimbriae 14 (SEF14) putative tip adhesin, SefD.     

Mechanisms of atrial-selective block of Na⁺ channels by ranolazine: II. Insights from a mathematical model

Block of Na(+) channel conductance by ranolazine displays marked atrial selectivity that is an order of magnitude higher that of other class I antiarrhythmic drugs. Here, we present a Markovian model of the Na(+) channel gating, which includes activation-inactivation coupling, aimed at elucidating the mechanisms underlying this potent atrial selectivity of ranolazine. The model incorporates experimentally observed differences between atrial and ventricular Na(+) channel gating, including a more negative position of the steady-state inactivation curve in atrial versus ventricular cells. The model assumes that ranolazine requires a hydrophilic access pathway to the channel binding site, which is modulated by both activation and inactivation gates of the channel. Kinetic rate constants were obtained using guarded receptor analysis of the use-dependent block of the fast Na(+) current (I(Na)). The model successfully reproduces all experimentally observed phenomena, including the shift of channel availability, the sensitivity of block to holding or diastolic potential, and the preferential block of slow versus fast I(Na.) Using atrial and ventricular action potential-shaped voltage pulses, the model confirms significantly greater use-dependent block of peak I(Na) in atrial versus ventricular cells. The model highlights the importance of action potential prolongation and of a steeper voltage dependence of the time constant of unbinding of ranolazine from the atrial Na(+) channel in the development of use-dependent I(Na) block. Our model predictions indicate that differences in channel gating properties as well as action potential morphology between atrial and ventricular cells contribute equally to the atrial selectivity of ranolazine. The model indicates that the steep voltage dependence of ranolazine interaction with the Na(+) channel at negative potentials underlies the mechanism of the predominant block of I(Na) in atrial cells by ranolazine.     

The effect of ATP-sensitive K+ channels on the electrical burst activity and insulin secretion in pancreatic β-cells

In recent years, the electrical burst activity of the insulin releasing pancreatic β-cells has attracted many experimentalists and theoreticians, largely because of its functional importance, but also because of the nonlinear nature of the burst activity. The ATP-sensitive K⁺ channels are believed to play an important role in electrical activity and insulin release. In this paper, we show by computer simulation how ATP and antidiabetic drugs can lengthen the plateau fraction of bursting and how these chemicals can increase the intracellular Ca²⁺ level in the pancreatic β-cell.     

Effect of cold adaptation of α-and β-adrenergic responses of blood pressure in hindlimb and small intestine in situ and systemic blood pressure in rabbits

Changes in the main parameters of α-and β-adrenergic responses, sensitivity to agonists (EC ₅₀) and maximum response (P _m) of hindlimb and small intestinal blood pressure in situ and systemic blood pressure were studied in rabbits adapted to cold for 1–30 days (daily exposures to ?10°C for 6 h). The responses to phenylephrine, noradrenaline, adrenaline, clonidine (α-agonists), and isopropylnoradrenaline (β-agonist) corresponded to the equation p = (P _m A ⁿ )/(EC ₅₀ ⁿ + A ⁿ ) (1) with n = 1 and n = 2, respectively. Cold adaptation induced reciprocal changes in the response of both EC ₅₀ and P _m to α-agonists and in the response of P _m alone to isopropylnoradrenaline. The significant differences of the parameters from control observed during the first 5 days of adaptation gradually decreased by day 30. After 10 days of adaptation, the efficiency (E = P _m/2EC ₅₀) of response to α-and β-agonists of adrenoceptors significantly increased.     

On the effect of sharp rises in blood pressure in the Shah–Humphrey model for intracranial saccular aneurysms

We consider the model originally proposed by Shah and Humphrey (J Biomech 32:593–599, 1999) for a class of intracranial saccular aneurysms and show that for constant pressure the addition of the viscoelastic term corresponding to the presence of cerebral spinal fluid outside the membrane, no matter how small, does ensure convergence to an equilibrium. Our arguments apply to a general equation of this type, and thus also hold for variations of this model such as that proposed by David and Humphrey (J Biomech 36:1143–1150, 2003). On the other hand, it is known that the presence of damping may destabilize periodic orbits of periodically forced systems or even prevent them from existing altogether. We present numerical simulations showing that for some forcing terms the high-frequency oscillations do not die out with time, and a much more complex behaviour may emerge as a discontinuous forcing term is approached. The key point for this situation to occur is related to the derivative of the forcing term, supporting the hypothesis that sharper rises (or falls) in blood pressure may increase the risk of aneurysm rupture.     

Trafficking-deficient hERG K⁺ channels linked to long QT syndrome are regulated by a microtubule-dependent quality control compartment in the ER

The human ether-a-go-go related gene (hERG) encodes the voltage-gated K(+) channel that underlies the rapidly activating delayed-rectifier current in cardiac myocytes. hERG is synthesized in the endoplasmic reticulum (ER) as an "immature" N-linked glycoprotein and is terminally glycosylated in the Golgi apparatus. Most hERG missense mutations linked to long QT syndrome type 2 (LQT2) reduce the terminal glycosylation and functional expression. We tested the hypothesis that a distinct pre-Golgi compartment negatively regulates the trafficking of some LQT2 mutations to the Golgi apparatus. We found that treating cells in nocodazole, a microtubule depolymerizing agent, altered the subcellular localization, functional expression, and glycosylation of the LQT2 mutation G601S-hERG differently from wild-type hERG (WT-hERG). G601S-hERG quickly redistributed to peripheral compartments that partially colocalized with KDEL (Lys-Asp-Glu-Leu) chaperones but not calnexin, Sec31, or the ER golgi intermediate compartment (ERGIC). Treating cells in E-4031, a drug that increases the functional expression of G601S-hERG, prevented the accumulation of G601S-hERG to the peripheral compartments and increased G601S-hERG colocalization with the ERGIC. Coexpressing the temperature-sensitive mutant G protein from vesicular stomatitis virus, a mutant N-linked glycoprotein that is retained in the ER, showed it was not restricted to the same peripheral compartments as G601S-hERG at nonpermissive temperatures. We conclude that the trafficking of G601S-hERG is negatively regulated by a microtubule-dependent compartment within the ER. Identifying mechanisms that prevent the sorting or promote the release of LQT2 channels from this compartment may represent a novel therapeutic strategy for LQT2.     

Epithelial Na⁺ sodium channels in magnocellular cells of the rat supraoptic and paraventricular nuclei

The epithelial Na? channels (ENaCs) are present in kidney and contribute to Na? and water homeostasis. All three ENaC subunits (α, β, and γ) were demonstrated in the cardiovascular regulatory centers of the rat brain, including the magnocellular neurons (MNCs) in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN). However, the functional significance of ENaCs in vasopressin (VP) and oxytocin (OT) synthesizing MNCs is completely unknown. In this study, we show with immunocytochemical double-labeling that the α-ENaC is colocalized with either VP or OT in MNCs in the SON and PVN. In addition, parvocellular neurons in the dorsal, ventrolateral, and posterior subregions of the PVN (not immunoreactive to VP or OT) are also immunoreactive for α-ENaC. In contrast, immunoreactivity to β- and γ-ENaC is colocalized with VP alone within the MNCs. Furthermore, immunoreactivity for a known target for ENaC expression, the mineralcorticoid receptor (MR), is colocalized with both VP and OT in MNCs. Using single-cell RT-PCR, we detected mRNA for all three ENaC subunits and MR in cDNA libraries derived from single MNCs. In whole cell voltage clamp recordings, application of the ENaC blocker benzamil reversibly reduced a steady-state inward current and decreased cell membrane conductance approximately twofold. Finally, benzamil caused membrane hyperpolarization in a majority of VP and about one-half of OT neurons in both spontaneously firing and quiet cells. These results strongly suggest the presence of functional ENaCs that may affect the firing patterns of MNCs, which ultimately control the secretion of VP and OT.     

Two sites for adenine-nucleotide regulation of ATP-sensitive potassium channels in mouse pancreatic β-cells and HIT cells

Summary ATP-inhibited potassium channels (K(ATP)) were studied in excised, inside-out patches from cultured adult mouse pancreatic -cells and HIT cells. In the absence of ATP, ADP opened K(ATP) channels at concentrations as low as 10 m and as high as 500 m, with maximal activation between 10 and 100 m ADP in mouse -cell membrane patches. At concentrations greater than 500 m, ADP inhibited K(ATP) channels while 10 mm virtually abolished channel activity. HIT cell channels had a similar biphasic response to ADP except that more than 1 mm ADP was required for inhibition. The channel opening effect of ADP required magnesium while channel inhibition did not. Using creatine/creatine phosphate solutions with creatine phosphokinase to fix ATP and ADP concentrations, we found substantially different K(ATP)-channel activity with solutions having the same ATP/ADP ratio but different absolute total nucleotide levels. To account for ATP-ADP competition, we propose a new model of channel-nucleotide interactions with two kinds of ADP binding sites regulating the channel. One site specifically binds MgADP and increases channel opening. The other, the previously described ATP site, binds either ATP or ADP and decreases channel opening. This model very closely fits the ADP concentration-response curve and, when incorporated into a model of -cell membrane potential, increasing ADP in the 10 and 100 m range is predicted to compete very effectively with millimolar levels of ATP to hyperpolarize -cells.The results suggest that (i) K(ATP)-channel activity is not well predicted by the ATP/ADP ratio, and (ii) ADP is a plausible regulator of K(ATP) channels even if its free cytoplasmic concentration is in the 10–100 m range as suggested by biochemical studies.We would like to thank Mr. Louis Stamps for expert technical assistance and Dr. Wil Fujimoto and Ms. Jeanette Teague for generously providing HIT cells obtained from Dr. Robert Santerre at Eli Lilly. We would also like to thank Dr. Michel Vivaudou for providing the program ALEX. Support was provided by the NIH and the Department of Veterans Affairs.     

The evolution of cardenolide-resistant forms of Na⁺,K⁺ -ATPase in Danainae butterflies

Cardenolides are a class of plant secondary compounds that inhibit the proper functioning of the Na⁺, K⁺‐ATPase enzyme in susceptible animals. Nonetheless, many insect species are able to sequester cardenolides for their own defence. These include butterflies in the subfamily Danainae (Family: Nymphalidae) such as the monarch (Danaus plexippus). Previous studies demonstrated that monarchs harbour an asparagine (N) to histidine (H) substitution (N122H) in the α subunit of Na⁺, K⁺‐ATPase (ATPα) that reduces this enzyme’s sensitivity to cardenolides. More recently, it has been suggested that at ATPα position 111, monarchs may also harbour a leucine (L)/glutamine (Q) polymorphism. This later amino acid could also contribute to cardenolide insensitivity. However, here we find that incorrect annotation of the initially reported DNA sequence for ATPα has led to several erroneous conclusions. Using a population genetic and phylogenetic analysis of monarchs and their close relatives, we show that an ancient Q111L substitution occurred prior to the radiation of all Danainae, followed by a second substitution at the same site to valine (V), which arose before the diversification of the Danaus genus. In contrast, N122H appears to be a recent substitution specific to monarchs. Surprisingly, examination of a broader insect phylogeny reveals that the same progression of amino acid substitutions (Q111L → L111V + N122H) has also occurred in Chyrsochus beetles (Family: Chrysomelidae, Subfamily: Eumolpinae) that feed on cardenolide‐containing host plants. The parallel pattern of amino acid substitution in these two distantly related lineages is consistent with an adaptive role for these substitutions in reducing cardenolide sensitivity and suggests that their temporal order may be limited by epistatic interactions.     

Association of plasma Aß peptides with blood pressure in the elderly

Background

Aß peptides are often considered as catabolic by-products of the amyloid ß protein precursor (APP), with unknown physiological functions. However, several biological properties have been tentatively attributed to these peptides, including a role in vasomotion.We assess whether plasma Aß peptide levels might be associated with systolic and diastolic blood pressure values (SBP and DBP, respectively).

Methodology/Principal Findings

Plasma Aß_1-40 and Aß_1-42 levels were measured using an xMAP-based assay in 1,972 individuals (none of whom were taking antihypertensive drugs) from 3 independent studies: the French population-based 3C and MONA-LISA (Lille) studies (n = 627 and n = 769, respectively) and the Australian, longitudinal AIBL study (n = 576). In the combined sample, the Aß_1-42/ Aß_1-40 ratio was significantly and inversely associated with SBP (p = 0.03) and a similar trend was observed for DBP (p = 0.06). Using the median age (69) as a cut-off, the Aß_1-42/Aß_1-40 ratio was strongly associated with both SBP and DBP in elderly individuals (p = 0.002 and p = 0.03, respectively). Consistently, a high Aß_1-42/ Aß_1-40 ratio was associated with a lower risk of hypertension in both the combined whole sample (odds ratio [OR], 0.71; 95% confidence interval [CI], 0.56-0.90) and (to an even greater extent) in the elderly subjects (OR, 0.53; 95% CI, 0.37–0.75). Lastly, all these associations appeared to be primarily driven by the level of plasma Aß_1-40.

Conclusion

The plasma Aß_1-42/Aß_1-40 ratio is inversely associated with SBP, DBP and the risk of hypertension in elderly subjects, suggesting that Aß peptides affect blood pressure in vivo. These results may be particularly relevant in Alzheimer''s disease, in which a high Aß_1-42/Aß_1-40 plasma ratio is reportedly associated with a decreased risk of incident disease.     

A substrate translocation trajectory in a cytoplasm-facing topological model of the monocarboxylate/H⁺ symporter Jen1p

Previous mutational analysis of Jen1p, a Saccharomyces cerevisiae monocarboxylate/H⁺ symporter of the Major Facilitator Superfamily, has suggested that the consensus sequence ³⁷⁹NXX[S/T]HX[S/T]QD³⁸⁷ in transmembrane segment VII (TMS‐VII) is part of the substrate translocation pathway. Here, we rationally design, analyse and show that several novel mutations in TMS‐V and TMS‐XI directly modify Jen1p function. Among the residues studied, F270 (TMS‐V) and Q498 (TMS‐XI) are critical specificity determinants for the distinction of mono‐ from dicarboxylates, and N501 (TMS‐XI) is a critical residue for function. Using a model created on the basis of Jen1p similarity with the GlpT permease, we show that all polar residues critical for function within TMS‐VII and TMS‐XI (N379, H383, D387, Q498, N501) are perfectly aligned in an imaginary axis that lies parallel to the protein pore. This model and subsequent mutational analysis further reveal that an additional polar residue facing the pore, R188 (TMS‐II), is irreplaceable for function. Our model also justifies the role of F270 and Q498 in substrate specificity. Finally, docking calculations reveal a ‘trajectory‐like’ substrate displacement within the Jen1p pore, where R188 plays a major dynamic role mediating the orderly relocation of the substrate by subsequent H‐bond interactions involving itself and residues H383, N501 and Q498.     

The diabetogenic agent alloxan increases K+ permeability by a mechanism involving activation of ATP-sensitive K+-channels in mouse pancreatic β-cells

The effects of the diabetogenic agent, alloxan, on membrane potential, input resistance and electrical activity of normal mouse pancreatic -cells were studied. Tetraethylammonium (TEA), quinine and Glyburide were used to block K⁺-channels and to elucidate the mechanisms underlying alloxan's effects on -cell membrane potential. Exposure of the islet to alloxan (75–100 M) in the presence of glucose (11 mM), produced a rapid (15 sec), transient inhibition of electrical activity, often accompanied by hyperpolarization of the membrane, and this was followed by recovery of the burst pattern. This early effect of alloxan was followed after approximately 15 min by a complete inhibition of electrical activity and hyperpolarization. The inhibition accompanied by hyperpolarization was associated with a decrease in input resistance, indicating increased K⁺-conductance. Both the transient and delayed effects of alloxan were blocked by glucose (33 mM), quinine and glyburide but not by other conditions which induced continuous electrical activity such as elevated external [K⁺] (10 mM), ouabain, K⁺ removal, or TEA (20 mM). The transient inhibition induced by alloxan may be due to a direct competition with glucose transport/metabolism since it did not occur when alpha-keto isocaproic acid (KIC) was used to induce electrical activity. The delayed inhibition may reflect indirect effects of accumulation of this agent or its metabolites within the cell. Since both effects of alloxan are blocked by glyburide they appear to involve activation of the ATP-sensitive K⁺-channel (K-ATP).     

Regulation of voltage-gated K channels by glucose metabolism in pancreatic β-cells

Regulation of delayed rectifier-type K⁺ channels (Kv-channels) by glucose was studied in rat pancreatic β-cells. The Kv-channel current was increased in amplitudes by increasing glucose concentration from 2.8 to 16.6 mM, while it was decreased by 2.8 mM glucose in a reversible manner (down-regulation) in both perforated and conventional whole-cell modes. The current was decreased by FCCP, intrapipette 0 mM ATP or AMPPNP. Glyceraldehyde, pyruvic acid, 2-ketoisocaproic acid, and 10 mM MgATP prevented the down-regulation induced by 2.8 mM or less glucose. The residual current after treatment with Kv2.1-specific blocker, guangxitoxin-1E, was unchanged by lowering or increasing glucose concentration. We conclude that glucose metabolism regulates Kv2.1 channels in rats β-cells via altering MgATP levels.     

Characterization of the Driving Force as a Modulator of Gating in Cardiac ATP-sensitive K+ Channels — Evidence for Specific Elementary Properties

Single cardiac ATP-sensitive K⁺ channels and, comparatively, two other members of the inwardly rectifying K⁺ channel family, cardiac K⁺ _(ir) and K⁺ _(ACh) channels, were studied in the inside-out recording mode in order to analyze influence and significance of the electrochemical K⁺ gradient for open-state kinetics of these K⁺ channels. The conductive state of K⁺ _(ATP) channels was defined as a function of the electrochemical K⁺ gradient in that increased driving force correlates with shortened open-channel lifetime. Flux coupling of gating can be largely excluded as the underlying mechanism for two reasons: (i) τ_open proved identical in 23 pS, 56 pS and 80 pS channels; (ii) K⁺ _(ATP) channel protonation by an external pH shift from 9.5 to 5.5 reduced conductance without a concomitant detectable change of τ_open. Since gating continued to operate at E _K , i.e., in the absence of K⁺ permeation through the pore, K⁺ driving force cannot be causally involved in gating. Rather the driving force acts to modulate the gating process similar to Rb⁺ whose interference with an externally located binding site stabilizes the open state. In K⁺ _(ir) and K⁺ _(ACh) channels, the open state is essentially independent on driving force meaning that their gating apparatus does not sense the electrochemical K⁺ gradient. Thus, K⁺ _(ATP) channels differ in an important functional aspect which may be tentatively explained by a structural peculiarity of their gating apparatus. Received: 24 March 1997/Revised: 24 April 1998