Short peptide constructs mimic agonist sites of AT(1)R and BK receptors.

Extracellular peptide ligand binding sites, which bind the N-termini of angiotensin II (AngII) and bradykinin (BK) peptides, are located on the N-terminal and extracellular loop 3 regions of the AT(1)R and BKRB(1) or BKRB(2) G-protein-coupled receptors (GPCRs). Here we synthesized peptides P15 and P13 corresponding to these receptor fragments and showed that only constructs in which these peptides were linked by S-S bond, and cyclized by closing the gap between them, could bind agonists. The formation of construct-agonist complexes was revealed by electron paramagnetic resonance spectra and fluorescence measurements of spin labeled biologically active analogs of AngII and BK (Toac(1)-AngII and Toac(0)-BK), where Toac is the amino acid-type paramagnetic and fluorescence quencher 2, 2, 6, 6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid. The inactive derivatives Toac(3)-AngII and Toac(3)-BK were used as controls. The interactions characterized by a significant immobilization of Toac and quenching of fluorescence in complexes between agonists and cyclic constructs were specific for each system of peptide-receptor construct assayed since no crossed reactions or reaction with inactive peptides could be detected. Similarities among AT, BKR, and chemokine receptors were identified, thus resulting in a configuration for AT(1)R and BKRB cyclic constructs based on the structure of the CXCR(4), an α-chemokine GPCR-type receptor.

Conformational properties of angiotensin II and its active and inactive TOAC-labeled analogs in the presence of micelles. Electron paramagnetic resonance, fluorescence, and circular dichroism studies.

The interaction between angiotensin II (AII, DRVYIHPF) and its analogs carrying 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) and detergents--negatively charged sodium dodecyl sulfate (SDS) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS)--was examined by means of EPR, CD, and fluorescence. EPR spectra of partially active TOAC1-AII and inactive TOAC3-AII in aqueous solution indicated fast tumbling, the freedom of motion being greater at the N-terminus. Line broadening occurred upon interaction with micelles. Below SDS critical micelle concentration, broader lines indicated complex formation with tighter molecular packing than in micelles. Small changes in hyperfine splittings evinced TOAC location at the micelle-water interface. The interaction with anionic micelles was more effective than with zwitterionic micelles. Peptide-micelle interaction caused fluorescence increase. The TOAC-promoted intramolecular fluorescence quenching was more pronounced for TOAC3-AII because of the proximity between the nitroxide and Tyr4. CD spectra showed that although both AII and TOAC1-AII presented flexible conformations in water, TOAC3-AII displayed conformational restriction because of the TOAC-imposed bend (Schreier et al., Biopolymers 2004, 74, 389). In HPS, conformational changes were observed for the labeled peptides at neutral and basic pH. In SDS, all peptides underwent pH-dependent conformational changes. Although the spectra suggested similar folds for AII and TOAC1-AII, different conformations were acquired by TOAC3-AII. The membrane environment has been hypothesized to shift conformational equilibria so as to stabilize the receptor-bound conformation of ligands. The fact that TOAC3-AII is unable to acquire conformations similar to those of native AII and partially active TOAC1-AII is probably the explanation for its lack of biological activity.

The angiotensin II AT1 receptor structure-activity correlations in the light of rhodopsin structure.

The most prevalent physiological effects of ANG II, the main product of the renin-angiotensin system, are mediated by the AT1 receptor, a rhodopsin-like AGPCR. Numerous studies of the cardiovascular effects of synthetic peptide analogs allowed a detailed mapping of ANG II's structural requirements for receptor binding and activation, which were complemented by site-directed mutagenesis studies on the AT1 receptor to investigate the role of its structure in ligand binding, signal transduction, phosphorylation, binding to arrestins, internalization, desensitization, tachyphylaxis, and other properties. The knowledge of the high-resolution structure of rhodopsin allowed homology modeling of the AT1 receptor. The models thus built and mutagenesis data indicate that physiological (agonist binding) or constitutive (mutated receptor) activation may involve different degrees of expansion of the receptor's central cavity. Residues in ANG II structure seem to control these conformational changes and to dictate the type of cytosolic event elicited during the activation. 1) Agonist aromatic residues (Phe8 and Tyr4) favor the coupling to G protein, and 2) absence of these residues can favor a mechanism leading directly to receptor internalization via phosphorylation by specific kinases of the receptor's COOH-terminal Ser and Thr residues, arrestin binding, and clathrin-dependent coated-pit vesicles. On the other hand, the NH2-terminal residues of the agonists ANG II and [Sar1]-ANG II were found to bind by two distinct modes to the AT1 receptor extracellular site flanked by the COOH-terminal segments of the EC-3 loop and the NH2-terminal domain. Since the [Sar1]-ligand is the most potent molecule to trigger tachyphylaxis in AT1 receptors, it was suggested that its corresponding binding mode might be associated with this special condition of receptors.

Functional rescue of a defective angiotensin II AT1 receptor mutant by the Mas protooncogene.

Earlier studies with Mas protooncogene, a member of the G-protein-coupled receptor family, have proposed this gene to code for a functional AngII receptor, however further results did not confirm this assumption. In this work we investigated the hypothesis that a heterodimeration AT(1)/Mas could result in a functional interaction between both receptors. For this purpose, CHO or COS-7 cells were transfected with the wild-type AT(1) receptor, a non-functional AT(1) receptor double mutant (C18F-K20A) and Mas or with WT/Mas and C18F-K20A/Mas. Cells single-expressing Mas or C18F/K20A did not show any binding for AngII. The co-expression of the wild-type AT(1) receptor and Mas showed a binding profile similar to that observed for the wild-type AT(1) expressed alone. Surprisingly, the co-expression of the double mutant C18F/K20A and Mas evoked a total recovery of the binding affinity for AngII to a level similar to that obtained for the wild-type AT(1). Functional measurements using inositol phosphate and extracellular acidification rate assays also showed a clear recovery of activity for AngII on cells co-expressing the mutant C18F/K20A and Mas. In addition, immunofluorescence analysis localized the AT(1) receptor mainly at the plasma membrane and the mutant C18F-K20A exclusively inside the cells. However, the co-expression of C18F-K20A mutant with the Mas changed the distribution pattern of the mutant, with intense signals at the plasma membrane, comparable to those observed in cells expressing the wild-type AT(1) receptor. These results support the hypothesis that Mas is able to rescue binding and functionality of the defective C18F-K20A mutant by dimerization.

Participation of transmembrane proline 82 in angiotensin II AT1 receptor signal transduction.

Most of the classical physiological effects of the octapeptide angiotensin II (AngII) are produced by activating the AT1 receptor which belongs to the G-protein coupled receptor family (GPCR). Peptidic GPCRs may be functionally divided in three regions: (i) extracellular domains involved in ligand binding; (ii) intracellular domains implicated in agonist-induced coupling to G protein and (iii) seven transmembrane domains (TM) involved in signal transduction. The TM regions of such receptors have peculiar characteristics such as the presence of proline residues. In this project we aimed to investigate the participation of two highly conserved proline residues (Pro82 and Pro162), located in TM II and TM IV, respectively, in AT1 receptor signal transduction. Both mutations did not cause major alterations in AngII affinity. Functional assays indicated that the P162A mutant did not influence the signal transduction. On the other hand, a potent deleterious effect of P82A mutation on signal transduction was observed. We believe that the Pro82 residue is crucial to signal transduction, although it is not possible to say yet if this is due to a direct participation or if due to a structural rearrangement of TM II. In this last hypothesis, the removal of proline residue might be correlated to a removal of a kink, which in turn can be involved in the correct positioning of residues involved in signal transduction.

Disruption of the kinin B1 receptor gene affects potentiating effect of captopril on BK-induced contraction in mice stomach fundus.

A transgenic mouse model, deficient in kinin B(1) receptor (B(1)(-/-)) was used to evaluate the role of B(2) receptor in the smooth muscle stomach fundus. The results showed that the potency of bradykinin (BK) to induce contraction in the gastric tissue was maintained whereas the efficacy was markedly reduced. The angiotensin converting enzyme (ACE) inhibitor captopril potentiated BK-induced effect in wild type (WT) but not in B(1)(-/-) fundus. However, ACE activity detected by the convertion of Ang I to Ang II was inhibited by captopril in both types of gastric tissues. Taking into account the hypothesis that captopril and ACE bind to the B(2) receptor, we suggest that this complex was not formed in the stomach deficient in B(1) receptor. Therefore, our finding strongly support the hypothesis that in smooth muscles that constitutively express the kinin B(1) and B(2) receptors, an interaction between captopril and ACE, B(1) and B(2) receptors should occur forming a complex protein interaction for the potentiating effect of ACE on kinin receptors.

Role of the kinin B1 receptor in insulin homeostasis and pancreatic islet function.

Kinins are potent vasoactive peptides generated in blood and tissues by the kallikrein serine proteases. Two distinct kinin receptors have been described, one constitutive (subtype B2) and one inducible (subtype B1), and many physiological functions have been attributed to these receptors, including glucose homeostasis and control of vascular permeability. In this study we show that mice lacking the kinin B1 receptor (B1-/- mice) have lower fasting plasma glucose concentrations but exhibit higher glycemia after feeding when compared to wild-type mice. B1-/- mice also present pancreas abnormalities, characterized by fewer pancreatic islets and lower insulin content, which leads to hypoinsulinemia and reduced insulin release after a glucose load. Nevertheless, an insulin tolerance test indicated higher sensitivity in B1-/- mice. In line with this phenotype, pancreatic vascular permeability was shown to be reduced in B1 receptor-ablated mice. The B1 agonist desArg9bradykinin injected intravenously can induce the release of insulin into serum, and this effect was not observed in the B1-/- mice or in isolated islets. Our data demonstrate the importance of the kinin B1 receptor in the control of pancreatic vascular homeostasis and insulin release, highlighting a new role for this receptor in the pathogenesis of diabetes and related diseases.

Role of the Cys18-Cys274 disulfide bond and of the third extracellular loop in the constitutive activation and internalization of angiotensin II type 1 receptor.

An insertion of residues in the third extracellular loop and a disulfide bond linking this loop to the N-terminal domain were identified in a structural model of a G-protein coupled receptor specific to angiotensin II (AT1 receptor), built in homology to the seven-transmembrane-helix bundle of rhodopsin. Both the insertion and the disulfide bond were located close to an extracellular locus, flanked by the second extracellular loop (EC-2), the third extracellular loop (EC-3) and the N-terminal domain of the receptor; they contained residues identified by mutagenesis studies to bind the angiotensin II N-terminal segment (residues D1 and R2). It was postulated that the insertion and the disulfide bond, also found in other receptors such as those for bradykinin, endothelin, purine and other ligands, might play a role in regulating the function of the AT1 receptor. This possibility was investigated by assaying AT1 forms devoid of the insertion and with mutations to Ser on both positions of Cys residues forming the disulfide bond. Binding and activation experiments showed that abolition of this bond led to constitutive activation, decay of agonist binding and receptor activation levels. Furthermore, the receptors thus mutated were translocated to cytosolic environments including those in the nucleus. The receptor form with full deletion of the EC-3 loop residue insertion, displayed a wild type receptor behavior.

A role for transmembrane domains V and VI in ligand binding and maturation of the angiotensin II AT1 receptor.

Several studies have proposed that angiotensin II (Ang II) binds to its receptor AT1 through interactions with residues in helices V and VI, suggesting that the distance between these helices is crucial for ligand binding. Based on a 3D model of AT1 in which the C-terminus of Ang II is docked, we identified the hydrophobic residues of TM V and VI pointing towards the external face of the helices, which may play a role in the structure of the binding pocket and in the structural integrity of the receptor. We performed a systematic mutagenesis study of these residues and examined the binding, localization, maturation, and dimerization of the mutated receptors. We found that mutations of hydrophobic residues to alanine in helix V do not alter binding, whereas mutations to glutamate lead to loss of binding without a loss in cell surface expression, suggesting that the external face of helix V may not directly participate in binding, but may rather contribute to the structure of the binding pocket. In contrast, mutations of hydrophobic residues to glutamate in helix VI lead to a loss in cell surface expression, suggesting that the external surface of helix VI plays a structural role and ensures correct folding of the receptor.

Ca2+-dependent K+ channels are targets for bradykinin B1 receptor ligands and for lipopolysaccharide in the rat aorta.

Although rat aorta smooth muscle cells in culture constitutively express bradykinin B1 receptors, the normotensive rat aorta does not respond to the bradykinin B1 receptor agonist des-Arg9-bradykinin, whereas vessels from the spontaneously hypertensive rat (SHR) respond to bradykinin B1 receptor agonists with cell membrane hyperpolarization and relaxation. Bacterial lipopolysaccharide also is inactive on the normotensive rat but hyperpolarizes the SHR aorta. To determine whether this could be due to the increased intracellular Ca2+ concentration ([Ca2+]i) in the SHR, we raised [Ca2+]i in normotensive rats by treatment with thapsigargin. In the thapsigargin-treated aorta, both lipopolysaccharide and des-Arg9-bradykinin induced hyperpolarization, which was reversed by the Ca2+-dependent K+ channel inhibitor iberiotoxin and by the bradykinin B1 receptor antagonists Lys-[Leu8]-des-Arg9-bradykinin and [Leu8]-des-Arg9-bradykinin. Thus the bradykinin B1 receptor, as well as lipopolysaccharide, needs activated Ca2+-dependent K+ channels for functional expression. The two bradykinin B1 receptor inhibitors, however, have effects on Ca2+-dependent K+ channels which are not mediated by bradykinin B1 receptors.

Molecular structure and transcriptional regulation by nuclear factor-kappaB of the mouse kinin B1 receptor gene.

Kinins are important mediators in cardiovascular homeostasis, inflammation, and nociception. Two kinin receptors have been described, B 1 and B 2 . The B 1 receptor is normally absent in healthy tissues, but is highly induced under pathological conditions. To understand the molecular mechanism of B 1 receptor up-regulation, we determined the mouse B 1 receptor gene structure, isolated and characterized the promoter region and studied its transcriptional regulation. The mouse B 1 receptor gene contains two exons (with the entire coding region located in the second exon) and a TATA-less promoter with multiple transcription start sites. A 7.7-kbp portion of the 5'-flanking region was examined for promoter activity in vascular smooth muscle cells (VSMCs). A minimal 92-bp fragment, located immediately upstream of the transcription start region, exerted basal and lipopolysaccharide (LPS)-inducible transcription activity in the sense and antisense orientation, and was thereby identified as an enhancer element. Nuclear extracts from VSMCs showed basal and LPS-inducible binding activity of nuclear factor (NF)-kappaB at this sequence. B 1 receptor transcription activation in response to LPS was abolished by cotransfection with IkappaBalphaDeltaN, an NF-kappaB repressor. In summary, our results reveal the structure of the mouse B 1 receptor gene and the involvement of NF-kappaB in the inducible mouse kinin B 1 receptor expression under pathological conditions.

Angiotensin II AT1 receptor mutants expressed in CHO cells caused morphological change and inhibition of cell growth.

To assess the importance of the leucine residues in positions 262 and 265 of the angiotensin AT(1) receptor for signaling pathways and receptor expression and regulation, we compared the properties of CHO cells transfected with the wild type or the L262D or L265D receptor point mutants. It was found that the two mutants significantly increased the basal intracellular cyclic AMP (cAMP) formation in an agonist-independent mode. The morphology transformation of CHO cells was correlated with the increased cAMP formation, since forskolin, a direct activator of adenylate cyclase mimicked this effect on WT-expressing CHO cells. DNA synthesis was found to be inhibited in these cell lines, indicating that cAMP may also have determined the inhibitory effect on cell growth, in addition to the cell transformation from a tumorigenic to a non-tumorigenic phenotype. However a role for an increased Ca2+ influx induced by the mutants in non-stimulated cells cannot be ruled out since this ion also was shown to cause transformed cells to regain the morphology and growth regulation.

Lys-[Leu8,des-Arg9]-bradykinin blocks lipopolysaccharide-induced SHR aorta hyperpolarization by inhibition of Ca(++)- and ATP-dependent K+ channels.

The mediators involved in the hyperpolarizing effects of lipopolysaccharide and of the bradykinin B1 receptor agonist des-Arg9-bradykinin on the rat aorta were investigated by comparing the responses of aortic rings of spontaneously hypertensive and normotensive Wistar rats. Endothelized rings from hypertensive rats were hyperpolarized by des-Arg9-bradykinin and lipopolysaccharide, whereas de-endothelized rings responded to lipopolysaccharide but not to des-Arg9-bradykinin. In endothelized preparations, the responses to des-Arg9-bradykinin were inhibited by Nomega-nitro-L-arginine and iberiotoxin. De-endothelized ring responses to lipopolysaccharide were inhibited by iberiotoxin, glibenclamide and B1 antagonist Lys-[Leu8,des-Arg9]-bradykinin. This antagonist also inhibited hyperpolarization by des-Arg9-bradykinin and by the á2-adrenoceptor agonist, brimonidine. Our results indicate that Ca(2+)-sensitive K+ channels are the final mediators of the responses to des-Arg9-bradykinin, whereas both Ca(2+)- and ATP-sensitive K+ channels mediate the responses to lipopolysaccharide. The inhibitory effects of Lys-[Leu8,des-Arg9]-bradykinin is due to a direct action on Ca(2+)- and ATP-sensitive potassium channels.

Conformational basis for the biological activity of TOAC-labeled angiotensin II and bradykinin: electron paramagnetic resonance, circular dichroism, and fluorescence studies.

N-Terminally and internally labeled analogues of the hormones angiotensin (AII, DRVYIHPF) and bradykinin (BK, RPPGFSPFR) were synthesized containing the paramagnetic amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC). TOAC replaced Asp1 (TOAC1-AII) and Val3 (TOAC3-AII) in AII and was inserted prior to Arg1 (TOAC0-BK) and replacing Pro3 (TOAC3-BK) in BK. The peptide conformational properties were examined as a function of trifluoroethanol (TFE) content and pH. Electron paramagnetic resonance spectra were sensitive to both variables and showed that internally labeled analogues yielded rotational correlation times (tauC) considerably larger than N-terminally labeled ones, evincing the greater freedom of motion of the N-terminus. In TFE, tauC increased due to viscosity effects. Calculation of tau(Cpeptide)/tau(CTOAC) ratios indicated that the peptides acquired more folded conformations. Circular dichroism spectra showed that, except for TOAC1-AII in TFE, the N-terminally labeled analogues displayed a conformational behavior similar to that of the parent peptides. In contrast, under all conditions, the TOAC3 derivatives acquired more restricted conformations. Fluorescence spectra of AII and its derivatives were especially sensitive to the ionization of Tyr4. Fluorescence quenching by the nitroxide moiety was much more pronounced for TOAC3-AII. The conformational behavior of the TOAC derivatives bears excellent correlation with their biological activity, since, while the N-terminally labeled peptides were partially active, their internally labeled counterparts were inactive [Nakaie, C. R., et al., Peptides 2002, 23, 65-70]. The data demonstrate that insertion of TOAC in the middle of the peptide chain induces conformational restrictions that lead to loss of backbone flexibility, not allowing the peptides to acquire their receptor-bound conformation.

Mutagenesis of the AT1 receptor reveals different binding modes of angiotensin II and [Sar1]-angiotensin II.

Homology modeling of the structure of the AT1 receptor, based on the high resolution rhodopsin crystal structure, indicated that it is unlikely that the binding of AngII to AT1 involves simultaneously all the receptor's residues reported in the literature to participate in this process. Site-directed mutagenesis using Ala substitution of charged residues Lys20, Arg23, Glu91 and Arg93 was performed to evaluate the participation of their side-chains in ligand binding and in triggering the cell's response. A comparative analysis by competition binding and functional assays using angiotensin II and the analog [Sar1]-angiotensin II suggests an important role for Arg23 of AT1 receptor in binding of the natural agonist. It is discussed whether some receptor's residues participate directly in the binding with AngII or whether they are part of a regulatory site.

Role of membrane potential and expression of endothelial factors in restenosis after angioplasty in SHR.

We examined the roles played by impaired K+ channels, diminished nitric oxide (NO) production, endothelin release, and smooth muscle membrane potential in the increased restenosis observed in spontaneously hypertensive rat (SHR) carotid arteries after angioplasty. The SHR carotid was found to be less polarized than that of normotensive Wistar rats (NWR), and it was further depolarized by the alpha2 agonist UK 14,304. This response was blocked by iberiotoxin, indicating that calcium-dependent K+ channels operate normally in the SHR carotid. Acetylcholine caused a hyperpolarization that was significantly smaller in SHR than in NWR carotids, indicating a deficient release of NO in the SHR. After angioplasty, SHR and NWR vessels were depolarized, returning to baseline after 10 days. In the SHR but not in the NWR the contralateral carotid was also depolarized, and this was prevented by the endothelin A/B receptor antagonist bosentan. After angioplasty, endothelin-1 plasma levels increased in both SHR and NWR, but the increase was significantly more prolonged in SHR. We found that the more pronounced restenosis observed in the SHR carotid after angioplasty is not due to impairment of calcium-dependent K+ channels but is related to the relatively depolarized vascular smooth muscles, involving endothelin release caused by reduced NO levels in that strain.

Angiotensin II-mediated cellular responses: a role for the 3'-untranslated region of the angiotensin AT1 receptor.

We have previously demonstrated that Chinese hamster ovary (CHO) cells transfected with the angiotensin II AT1 receptor gene containing only the coding region, presented tachyphylaxis to the total inositol phosphate (InsPs) and Ca2+ responses mediated by angiotensin II and [2-lysine]angiotensin II ([Lys2]angiotensin II). Now we have evaluated the possible role of the 3'-untranslated region of the angiotensin AT1 receptor mRNA in modulating the angiotensin AT1 receptor-mediated cellular responses. The binding parameters, as well as the Ca2+ and InsPs responses induced by angiotensin II and [Lys2]angiotensin II were similar in cells transfected with the angiotensin AT1 receptor with or without the 3'-untranslated region sequence. In cells transfected with the receptor containing the 3'-untranslated region sequence, angiotensin II-induced Ca2+ and InsPs responses were desensitized by repeated stimulations, whereas [Lys2]angiotensin II caused desensitization of InsPs production but not of Ca2+ uptake in these cells. Our results suggest that the 3'-untranslated region plays a role in modulating cell signalling involved in the tachyphylaxis of angiotensin AT1 receptor-mediated Ca2+ responses.

Identification of functionally conserved residues with the use of entropy-variability plots.

We introduce sequence entropy-variability plots as a method of analyzing families of protein sequences, and demonstrate this for three well-known sequence families: globins, ras-like proteins, and serine-proteases. The location of an aligned residue position in the entropy-variability plot correlates with structural characteristics, and with known facts about the roles of individual amino acids in the function of these proteins. The large numbers of known sequences in these families allowed us to introduce new filtering methods for variability patterns. The results are discussed in terms of a simple evolutionary model for functional proteins.

Sequence analysis reveals how G protein-coupled receptors transduce the signal to the G protein.

Sequence entropy-variability plots based on alignments of very large numbers of sequences-can indicate the location in proteins of the main active site and modulator sites. In the previous article in this issue, we applied this observation to a series of well-studied proteins and concluded that it was possible to detect most of the residues with a known functional role. Here, we apply the method to rhodopsin-like G protein-coupled receptors. Our conclusion is that G protein binding is the main evolutionary constraint on these receptors, and that other ligands, such as agonists, act as modulators. The activation of the receptors can be described as a simple, two-step process, and the residues involved in signal transduction can be identified.

Characterization of alpha2-adrenoceptors in smooth muscles of the spontaneously hypertensive rat aorta.

Previous works have shown that the alpha(2)-adrenoceptor agonist UK 14,304 induced the relaxation and hyperpolarization of the rat aorta, mediated by alpha(2)-adrenoceptors present in the smooth muscles, through small-conductance, ATP-sensitive K(+) channels. We now report that in spontaneously hypertensive rat (SHR) aortic rings, UK 14,304 induced concentration-dependent hyperpolarizing responses, which were inhibited by yohimbine, an alpha(2)-adrenoceptor inhibitor, and by glibenclamide, a specific inhibitor of small-conductance, ATP-sensitive K(+) channels. The responses were also partially inhibited by iberiotoxin and by apamin. Treatment with N(omega)-nitro-L-arginine (L-NNA) did not affect the response to UK 14,304. These results indicate that alpha(2)-adrenoceptors are present in SHR aortic smooth muscle cell membranes, but differ from those of normotensive animals regarding the K(+) channels involved in their responses. Moreover, the resting membrane potential (RMP) was significantly more negative in SHR than in normotensive rats. This relative hyperpolarized state is probably due to Ca(2+)-dependent K(+) channels being constitutively open in SHR, since the addition of iberiotoxin caused a significant depolarization of the aortic smooth muscle membranes in this strain.