Biomedical subjects

E Krause

Publications and source records attributed to E Krause.

At least 73 records · Page 4Linked to original sources

PubMed1997

Frontal-complex partial status epilepticus misdiagnosed as bipolar affective disorder in a 75-year-old man.

The incidence and prevalence of epilepsy increases with age, with the majority of cases having a known cause, and approximately half of elderly patients with epilepsy experiencing complex partial seizures that often present initially as neuropsychiatric symptoms. This presentation often delays diagnosis of the epileptiform disorder. To illustrate, we present the case of a 75-year-old man who was initially misdiagnosed with bipolar affective disorder later to be revealed as a frontal lobe seizure disorder.

Aged↗

PubMed1996

Depletion of intracellular calcium stores activates a calcium conducting nonselective cation current in mouse pancreatic acinar cells.

Receptor-mediated Ca2+ release from inositol (1,4,5)-trisphosphate (IP3)-sensitive Ca2+ stores causes "capacitative calcium entry" in many cell types (Putney, J. W., Jr. (1986) Cell Calcium 7, 1-12; Putney, J. W., Jr. (1990) Cell Calcium 11, 611-624). We used patch-clamp and fluorescence techniques in isolated mouse pancreatic acinar cells to identify ion currents and cytosolic calcium concentrations under conditions in which intracellular Ca2+ stores were emptied. We found that depletion of Ca2+ stores activated a calcium-release-activated nonselective cation current (ICRANC) which did not discriminate between monovalent cations. ICRANC possessed a significant conductance for Ca2+ and Ba2+. It was not inhibited by La3+, Gd3+, Co2+, or Cd2+ but was completely abolished by flufenamic acid or genistein. In whole cell and cell-attached recordings, a 40-45 pS nonselective cation channel was identified which was activated by Ca2+ store depletion. Calcium entry as detected by single cell fluorescence measurements with fluo-3 or fura-2, showed the same pharmacological properties as ICRANC. We conclude that in mouse pancreatic acinar cells 40-45 pS nonselective cation channels serve as a pathway for capacitative Ca2+ entry. This entry pathway differs from the previously described ICRAC (Hoth, M., and Penner, R. (1992) Nature 355, 353-356) in its ion-selectivity, pharmacological profile, and single-channel conductance.

Acetylcholine↗

PubMed1996

Peptide helicity and membrane surface charge modulate the balance of electrostatic and hydrophobic interactions with lipid bilayers and biological membranes.

An amphipathic model peptide, KLALKLALKALKAAKLA-NH2, and its complete double D-amino acid replacement set was used to analyze the process of peptide binding at lipid vesicles of different surface charge and to determine the structure of the lipid-bound peptides using CD spectroscopy. The relationship between peptide helicity, model membrane permeability, and biological activity has been studied by dye release from liposomes and investigation of antibacterial and hemolytic activity. The accumulation of cationic KLAL peptides at and the membrane-disturbing effect on bilayers of high negative surface charge were found to be dominated by charge interactions. Independent of any structural propensity, the cationic peptide side chains bind to the anionic phosphatidylglycerol moieties. The charge interactions hold the peptides at the bilayer surface, where they may disturb preferentially lipid headgroup organization by formation of peptide-lipid clusters. In contrast, KLAL peptide interaction with bilayers of low negative surface charge is highly dependent on peptide helicity. With decreasing amounts of anionic phosphatidylglycerol in the bilayer the membrane-disturbing effect of KLAL and other helical analogs substantially increases despite drastically reduced binding affinity. Less helical peptides exhibit reduced bilayer-disturbing activity, showing that the hydrophobic helix domain is decisive for binding at and inducing permeability in membranes of low negative surface charge. It is suggested that hydrophobic interactions drive the penetration of the amphipathic peptide structure into the inner membrane region, thus disturbing the arrangement of the lipid acyl chains and causing local disruption. On the basis of the proposed model for membrane disturbance, interactions modulating antibacterial and hemolytic activity are discussed.

Amino Acid Sequence↗

PubMed1996

Conformational and functional study of magainin 2 in model membrane environments using the new approach of systematic double-D-amino acid replacement.

Systematic double-D-amino acid replacement of adjacent amino acids has been used to study the secondary structure of the amphiphilic, antibiotic peptide magainin 2 amide (M2a) by circular dichroism spectroscopy. Bound to liposomes, the secondary structure of the peptide is characterized by a weak alpha-helix in the N-terminus and a stable alpha-helix between residues 9 and 21. The lack of conformational differences in the peptide when bound to vesicles of varying negative charge density indicates marked independence of the structure from electrostatic forces. The similarity of the helicity profiles observed for double D-isomers bound to vesicles and in the presence of sodium dodecyl sulfate micelles (SDS) clearly shows that SDS can mimic magainin-lipid interactions. In contrast, in 1:1 trifluoroethanol/buffer (v/v), the peptide exhibits a weak alpha-helix extended from the N- to the C-terminus. Dye release experiments from vesicles of phosphatidylglycerol showed that double-D-amino acid substitution only in the region of the stable helix results in a reduction of the membrane-permeabilizing ability. On vesicles with a reduced amount of acidic phospholipids, double-D-amino acid substitution in any position leads to a drastic reduction of peptide-induced membrane permeabilization. Whereas the activity of M2a on phosphatidylglycerol was found to be mainly electrostatically determined, hydrophobic interactions play a decisive role in the interaction with vesicles of reduced negative charge density. Fluorescence investigations of tryptophan-containing analogs of high and low helicity showed that differences in the location of the chromophores of the membrane-bound peptides do not exist.

Amides↗

PubMed1996

A single-point slight alteration set as a tool for structure-activity relationship studies of ovine corticotropin releasing factor.

In order to determine which amino acid side chains of ovine corticotropin releasing factor (oCRF) are most sensitive to alterations with respect to receptor binding and activation, we synthesized a single-point replacement set by replacing each residue by a similar, preferably proteinogenic amino acid, maintaining a minimal change of character at each position (Ser by Thr, Gln by Asn, Glu by Asp, Arg by Lys, and vice versa, Pro by N-MeAla, Ile by Leu, Leu by Nle, Phe by Trp, His by Ala, Val by Leu, Met by Nle, Ala by Leu). In general, any loss in the biological potency by a single-point substitution in oCRF parallels a decrease in receptor binding, indicating that, in contrast to previous suggestions, there is no specific side chain in the peptide that is more responsible for receptor activation than for receptor binding. In addition to Arg(16), Ala(31), and Arg(35), amino acid residues in the N-terminal sequence (5-14) were found to be sensitive to alteration, demonstrating their particular importance for the receptor interaction of CRF agonists. Most of the analogs tested exhibited agonistic potencies in an in vitro pituitary cell culture assay at a concentration of 0.3 nM, and all analogs showed full agonistic potency at 1 microM. In contrast to the results of an alanine replacement study, the strongest decrease in receptor binding and biological potency was observed for analogs with substitutions of hydrophilic amino acids Ser(7), Arg(16), Glu(17), or Asn(34). In the case of Ser(7) and Arg(16), side chain specific interactions with the receptor may be required for high affinity. Alanine replacements at positions 17 or 34 resulted in analogs that were as potent as oCRF, while replacement of Glu(17) by Asp or Asn(34) by Gln caused a dramatic loss in potency, thereby suggesting an important effect at sterically or conformationally sensitive positions. In contrast to corresponding alanine analogs which exhibited a significant loss in biological potency, slight alterations of lipophilic side chains at positions 6, 12, or 38 did not cause a significant reduction of receptor binding and activation, indicating that it is not specific side chains but rather lipophilicity which is essential at these positions. Indeed, replacement of Phe(12) by Trp provides an agonist with significantly increased receptor binding and biological potency.

Adrenocorticotropic Hormone↗

PubMed1996

Caged compounds of hydrolysis-resistant analogues of cAMP and cGMP: synthesis and application to cyclic nucleotide-gated channels.

Photolabile compounds which rapidly release cAMP or cGMP after photolysis are widely used for in situ studies of signaling pathways inside cells. We synthesized two novel caged compounds, 4,5-dimethoxy-2-nitrobenzyl 8-Br-cAMP (caged 8-Br-cAMP) and 4,5-dimethoxy-2-nitrobenzyl 8-Br-cGMP caged 8-BR-cGMP), which respectively release the hydrolysis-resistant analogues 8-Br-cAMP and 8-Br-cGMP. Their usefulness for physiological studies was examined in a mammalian cell line expressing the cyclic nucleotide-gated (CNG) ion channel of bovine olfactory sensory neurons. The synthesis procedure resulted in diastereomeric mixtures which were chromatographically separated into the axial and equatorial isomers of caged 8-BR-cAMP and of caged 8-BR-cGMP. The axial isomers which have a higher solubility and better solvolytic stability than the equatorial forms were used for experiments with CNG channels. Flashes of UV light produced steps in the concentration of 8-Br-cGMP which activated currents through CNG channels. Concentration steps inside the cell could be calibrated precisely using the relation between the ligand concentration and the normalized current. Similar results were obtained with caged 8-Br-cAMP. Control experiments with caged cGMP showed that flash-induced currents decayed within a few minutes because photoreleased cGMP was degraded by endogenous phosphodiesterase activity. The rise time of the 8-Br-cGMP-activated whole-cell current was consistent with a bimolecular reaction between channel and ligand.

1-Methyl-3-isobutylxanthine↗

PubMed1996

Assignment of the helical structure in neuropeptide Y by HPLC studies of methionine replacement analogues and 1H-NMR spectroscopy.

The HPLC retention behavior of three complete single methionine and methionine sulfoxide replacement sets of two 18-mer model peptides and neuropeptide Y (NPY) were investigated. All peptides were prepared by multiple solid-phase peptide synthesis. Plotting the retention time differences between methionine and methionine sulfoxide analogues vs the position of replacement shows that potentially alpha-helical peptides become helical on binding during reversed-phase high performance liquid chromatography. In the case of an amphipathic alpha-helix, the retention time differences change periodically with a 3-4 repeat pattern, which allow the location of amphipathic helical structures. Replacements in nonamphipathic alpha-helical domains cause local preferential binding areas and lead to sequence-dependent retention time profiles. Methionine replacement studies of NPY suggest an unstructured or extended conformation from Tyr1 to Ala12 connected to a well-defined amphipathic alpha-helix from Pro13 to Arg35. The assignment is confirmed by comparison of nuclear Overhauser effects based two-dimensional 1H-nmr spectroscopy and utilization of the C alpha H shift index method in 50% trifluoroethanol/50% water.

Amino Acid Sequence↗

PubMed1996

Temperature coefficients of amide proton NMR resonance frequencies in trifluoroethanol: a monitor of intramolecular hydrogen bonds in helical peptides.

2D 1H NMR spectroscopy of two alpha-helical peptides which differ in their amphipathicity has been used to investigate the relationships between amide-proton chemical shifts, amide-proton exchange rates, temperature, and trifluoroethanol (TFE) concentration. In 50% TFE, in which the peptides are maximally helical, the amide-proton chemical shift and temperature coefficient patterns are very similar to each other in each peptide. Temperature coefficients from -10 to -6 ppb/K, usually indicative of the lack of intramolecular hydrogen bonds, were observed even for hydrophobic amino acids in the center of the alpha-helices. However, slow hydrogen isotope exchange for residues from 4 to 16 in both 18-mer helices indicates intact intramolecular hydrogen bonds over most of the length of these peptides. Based on these anomalous observations, we suggest that the pattern of amide-proton shifts in alpha-helices in H20/TFE solvents is dominated by bifurcated intermolecular hydrogen-bond formation between the backbone carbonyl groups and TFE. The amide-proton chemical shift changes with increasing temperature may be interpreted by a disruption of intermolecular hydrogen bonds between carbonyl groups and the TFE in TFE/water rather than by the length of intramolecular hydrogen bonds in alpha-helices.

Hydrogen↗

PubMed1996

Numerical simulation of the blood flow in the human cardiovascular system.

This paper describes a numerical model of the human cardiovascular system. The model is composed of 15 elements connected in series representing the main parts of the system. Each element is composed of a rigid connecting tube and an elastic reservoir. The blood flow is described by a one-dimensional time-dependent Bernoulli equation. The action of the ventricles is simulated with a Hill's three-element model, adapted for the left and right heart. The closing of the four heart valves is simulated with the aid of time-dependent drag coefficients. Closing is achieved by letting the drag coefficient approach infinity. The resulting system of 32 non-linear ordinary differential equations is solved numerically with the Runge-Kutta method. The results of the simulation (pressure-time and volume-time dependence for the atria and ventricles and pressure forms in the aorta at a heart rate of 70 beats per minute) agree with the physiological data given in the literature. The model's input aortic impedance is 31.5 dyn s cm-5 which agrees with literature data given for aortic input impedance in man 26-80 dyn s cm-5). Long-term stability of the system was achieved. The cardiovascular system presented here can also be simulated at higher and varying heart rates--up to 200 beats per minute. The results of calculations for some pathological changes (e.g. valvular abnormalities) are discussed.

Algorithms↗

PubMed1996

Cardiovascular effects of conventional sulfonylureas and glimepiride.

Sulfonylureas have, in the past, been reported to have adverse cardiovascular effects. Glimepiride is a new sulfonylurea. In spite of stimulating less insulin secretion, it has, depending on the species, equal or higher blood glucose decreasing activity and according to preliminary studies less cardiovascular activity than glibenclamide. Further studies were performed to confirm the lower cardiovascular activity of glimepiride. The IC50 for inhibition of rilmakalim-activated KATP channel currents in isolated ventricular myocytes was 31.6 nM for glimepiride and 6.8 nM for glibenclamide. In endotoxin shock-rats at a dose of 1 x 2 mg/kg i.v., glibenclamide induced a significantly higher blood pressure increase than glimepiride. At two i.v. doses of 20 mg/kg 4 min apart, in normal rats, glibenclamide produced signs of ischemia in the ECG in nearly all animals, glimepiride almost none, in diabetic rats, glibenclamide produced in all animals a lethal cardiogenic shock preceeded by serious ECG changes, glimepiride only in one fifth of the animals. In open-chest dogs, on intracoronary infusion of equieffective blood glucose-lowering doses, glibenclamide, gliclazide and glimepiride all reduced coronary blood flow, increased coronary resistance, depressed the mechanical activity of the heart, enhanced myocardial O2-extraction, reduced the serum potassium level and induced a moderate endocardial ST-segment elevation, but glimepiride to a significantly less extent than glibenclamide and gliclazide. The presented data confirm that glimepiride at equivalent blood glucose decreasing doses has less cardiovascular activity than conventional sulfonylureas.

Animals↗

PubMed1996

Conformation of a water-soluble beta-sheet model peptide. A circular dichroism and Fourier-transform infrared spectroscopic study of double D-amino acid replacements.

Among peptide secondary structures beta-sheet domains have been much less intensively studied than alpha-helical conformations, mainly because of the lack of well characterized model peptides. In the present paper the secondary structure of a water-soluble de novo peptide consisting of 26 amino acids (DPKGDPKGVTVTVTVTVTGKGDPKPD-NH2) and the corresponding double D-amino acid replacement set have been studied by circular dichroism and Fourier-transform infrared spectroscopy. The model peptide was found to be unstructured in aqueous solution at peptide concentrations < 10(-3) mol/L but to adopt a predominantly beta-sheet structure in the presence of 15 mM sodium dodecyl sulfate or at apolar/water interfaces. Although the peptide is composed of amino acids with low helical propensity, it formed a single-stranded helical structure in aqueous trifluoroethanol. The D-amino acid replacement set was synthesized in order to study the conformational stability of the model peptide selectively in distinct regions. The data show that both the alpha-helix present in 50% trifluoroethanol as well as the beta-sheet domain formed in the presence of sodium dodecyl sulfate or at apolar/water interfaces, are located in the region between Val9 and Thr18. Pairwise substitution of adjacent amino acids by their corresponding D-amino acids provides a pronounced beta-sheet disturbance. These findings demonstrate that double D-amino acid replacements may be used to locate beta-sheet domains in peptides.

Amino Acid Sequence↗

PubMed1996

Structure activity studies of mast cell activation and hypotension induced by neuropeptide Y (NPY), centrally truncated and C-terminal NPY analogues.

1. Neuropeptide-induced histamine release is thought to occur via receptor-independent mechanisms, with net charge and lipophilicity being important factors. 2. In this study, the histamine releasing ability of neuropeptide Y (NPY), two C-terminal segments of NPY and 13 centrally truncated NPY analogues was examined. These results were compared with the ability of the peptides to bind to the Y2 receptor in the rabbit kidney membrane model and with their hypotensive actions in the anaesthetized-rat model. 3. All analogues tested, with the exception of [Glu4,25,33,35]-NPY(1-4)-Ahx-(25-36) and [Asp4,25,33,35]NPY(1-4)-Ahx-(25-36) which were devoid of histamine releasing activity, evoked a dose-dependent histamine release but there were marked differences between the peptides. The native peptide was the least active. 4. Histamine release was not linked to the ability of the peptides to displace NPY from Y2 receptors. There was a statistical correlation between the hypotensive effects expressed as ED10 values (mumol kg-1, which induced a blood pressure decrease of 10 mmHg) and the EC25 for histamine release (r = 0.62, P = 0.04), although histamine release may not be the sole determinant of the alterations in blood pressure. 5. There was a strong negative correlation between EC25 for histamine release and net positive charge (r = -0.93, P = 5.7 x 10(-7), i.e. increasing the net positive charge caused greater histamine release. However, there was a 12 fold difference in activity amongst the most positively charged analogues (+5). Helicity did not correlate with histamine releasing ability. 6. In the development of NPY-related drugs the avoidance of compounds with net positive charge is recommended.

Animals↗

PubMed1995

Influence of alpha-helicity, amphipathicity and D-amino acid incorporation on the peptide-induced mast cell activation.

Mast cell activation by polycationic substances is believed to result from a direct activation of G protein alpha subunits and it was suggested that the adaption of amphipathic, alpha-helical conformations would allow the peptide to reach the cytosolic compartment to interact with G proteins (Mousli et al., 194, Immunopharmacology 27, 1, for review). We investigated the histamine-releasing activity of model peptides as well as analogues of magainin 2 amide and neuropeptide Y with different amphipathicities and alpha-helix content on rat peritoneal mast cells. Amphipathic helicity is not a prerequisite for mast cell activation. Moreover, non-helical magainin peptides with high histamine-releasing activity were less active in the liberation of carboxyfluoresceine from negatively charged liposomes, indicating that peptide-induced mast cell activation and peptide-induced membrane perturbation do not correlate. In contrast to the negligible influence of the secondary structure, amino acid configuration may exert a striking influence on peptide-induced mast cell activation. Thus histamine-release by substance P was markedly impaired when the L-amino acids in the positively charged N-terminal region were replaced by D-amino acids, with [D-Arg1)substance P being the most inactive substance P diastreoisomer.

Amino Acid Sequence↗

PubMed1995

Structure effects of double D-amino acid replacements: a nuclear magnetic resonance and circular dichroism study using amphipathic model helices.

D-Amino acid replacements and the determination of resulting structural changes are a useful tool to recognize amphipathic helices in biologically active peptides such as neuropeptide Y and corticotropin-releasing factor. In this paper the secondary structures of one amphipathic alpha-helical peptide and its double D-amino acid analog have been determined by means of 1H NMR and CD spectroscopies under equivalent conditions. The chemical shifts (NH and C alpha H) and the analysis of nuclear Overhauser effects show a split of the continuous helix for the all-L peptide into two helices at the position of double D-amino acid replacement. Hydrogen exchange rates correlate with water accessibilities in the hydrophobic/hydrophilic face and confirm the amphipathic helical structure in the all-L peptide as well as in its double D-amino acid analog. A significantly accelerated hydrogen isotope exchange rate is observed for the D-Ala9 backbone proton, implying an increased flexibility at that position. These results show that the incorporation of an adjacent pair of D-amino acids only causes a local change in structure and flexibility, which makes the double D replacement interesting as a tool for specific helix-disturbing modifications to search for helical conformations in biologically active peptides.

Amino Acid Sequence↗

PubMed1995

Location of an amphipathic alpha-helix in peptides using reversed-phase HPLC retention behavior of D-amino acid analogs.

The reversed-phase HPLC retention behavior of D-amino acid replacement sets of an amphipathic model peptide, neuropeptide Y, and corticotropin releasing factor has been studied. The results demonstrate that D-amino acid substitutions destabilized the amphipathic alpha-helix, leading to a decrease of fractional helicity as determined by circular dichroism. The effect is enhanced by substitution of two adjacent D-amino acids and correlates well with a decrease of hydrophobic interaction during reversed-phase HPLC, caused by disturbance of the preferred binding domain of the stationary phase-bound peptide. In contrast, D-amino acid substitutions in nonamphipathic or disordered regions of peptides do not influence the retention time to the same extent. Thus, the "retention profile" that results from plotting the retention time vs the position of the double D-amino acid replacements provides an indication of the presence and location of an amphipathic alpha-helical secondary structure in peptides.

Amino Acid Sequence↗

PubMed1995

Recognition of alpha-helical peptide structures using high-performance liquid chromatographic retention data for D-amino acid analogues: influence of peptide amphipathicity and of stationary phase hydrophobicity.

The reversed-phase HPLC behaviour of double D-amino acid replacement sets of amphipathic and non-amphipathic helix-forming peptides consisting exclusively of leucine, lysine and alanine residues was studied on different polymer-encapsulated silica-based stationary phases. Plotting the retention times versus the position of D-amino acid substitution gives a characteristic pattern showing decreased retention times in the helical region. The retention time profile obtained using an amphipathic alpha-helix is caused by disturbance of the preferred binding domain of the stationary phase-bound peptide. However, the effect is similar but less pronounced using a non-amphipathic helical peptide that is unable to interact by a preferred binding site. The results demonstrate that reversed-phase HPLC data for peptide analogues provide an indication event of a non-amphipathic helical structure in peptides.

Amino Acid Sequence↗

PubMed1995

MALDI-MS for C-terminal sequence determination of peptides and proteins degraded by carboxypeptidase Y and P.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used for C-terminal amino acid sequence determination of peptides and proteins. The usefulness of MALDI-MS was demonstrated by analyzing peptide mixtures (C-terminal peptide ladder) which were generated by enzymatic digestion of substance P, glucagon, angiotensinogen, insulin B chain and myoglobin with the exopeptidases carboxypeptidase Y and P. The results clearly show that up to 11 amino acid residues can be determined in the pmol range by analyzing the molecular masses of the truncated peptides. For proteins it is possible to investigate enzymatic or chemical digests in the same manner.

Amino Acid Sequence↗

PubMed1995

Adenosine triphosphate-dependent K currents activated by metabolic inhibition in rat ventricular myocytes differ from those elicited by the channel opener rilmakalim.

Adenosine triphosphate (ATP) dependent potassium channels (KATP channels) in heart ventricular muscle cells can be activated by depletion of intracellular ATP stores as well as by channel openers. In the present study we examined whether properties of KATP channels are dependent on the mode of activation. Whole-cell and single-channel currents were investigated by use of the patch-clamp technique in isolated ventricular rat myocytes. The channel opener rilmakalim dose dependently activated whole-cell currents [concentration for half-maximal activation (EC50) = 1.1 microM, Hill coefficient = 3.1, saturation concentration 10 microM]. Metabolic inhibition with 2-deoxy-D-glucose (10 mmol/l) also activated KATP currents after a time lag of several minutes. These currents were about two-fold higher than the rilmakalim-activated currents (rilmakalim-activated current 3.9 +/- 0.2 nA, 2-deoxy-D-glucose-activated current 8.1 +/- 0.9 nA; both recorded at 0 mV clamp potential). While the rilmakalim-activated current could be blocked completely and with high affinity by the sulphonylurea glibenclamide [concentration for half-maximal inhibition (IC50) = 8 nM, Hill coefficient = 0.7] the 2-deoxy-D-glucose-activated current could only be blocked partially (by maximally 46%) and higher glibenclamide concentrations were needed (IC50 = 480 nM, Hill coefficient = 0.8). The partial loss of blocking efficiency after metabolic inhibition was not restricted to glibenclamide but was also observed with the sulfonylureas glimepiride and HB 985, as well as with the non-sulfonylureas HOE 511 and 5-hydroxy-decanoate. Single-channel studies were in accordance with these whole-cell experiments. Both rilmakalim and metabolic inhibition with the uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) activated single channels in the attached mode, where the number of current levels was significantly higher in the case of FCCP. Rilmakalim-activated channels were completely blocked by 10 microM glibenclamide, whereas several single-channel levels appeared in the presence of 100 microM glibenclamide after metabolic inhibition. In conclusion, after metabolic inhibition the amplitude of the activated KATP current is about twice as high as under saturating concentrations of the opener rilmakalim. Moreover, channels activated by metabolic inhibition lost part of their sensitivity to known channel blockers.

Adenosine Triphosphate↗