Maturation of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in 3D collagen matrix: Effects of niche cell supplementation and mechanical stimulation.

Cardiomyocytes derived from human embryonic stem cells (hESC-CMs) are regarded as a promising source for regenerative medicine, drug testing and disease modeling. Nevertheless, cardiomyocytes are immature in terms of their contractile structure, metabolism and electrophysiological properties. Here, we fabricate cardiac muscle strips by encapsulating hESC-CMs in collagen-based biomaterials. Supplementation of niche cells at 3% to the number of hESC-CMs enhance the maturation of the hESC-CMs in 3D tissue matrix. The benefits of adding mesenchymal stem cells (MSCs) are comparable to that of adding fibroblasts. These two cell types demonstrate similar effects in promoting the compaction and cell spreading, as well as expression of maturation markers at both gene and protein levels. Mechanical loading, particularly cyclic stretch, produces engineered cardiac tissues with higher maturity in terms of twitch force, elastic modulus, sarcomere length and molecular signature, when comparing to static stretch or non-stretched controls. The current study demonstrates that the application of niche cells and mechanical stretch both stimulate the maturation of hESC-CMs in 3D architecture. Our results therefore suggest that this 3D model can be used for in vitro cardiac maturation study. STATEMENT OF SIGNIFICANCE: Cardiomyocytes derived from human embryonic stem cells (hESC-CMs) are regarded as being a promising source of cells for regenerative medicine, drug testing and disease modeling. Nevertheless, cardiomyocytes are immature in terms of their contractile structure, metabolism and electrophysiological properties. In the current study, we have fabricated cardiac muscle strips by encapsulating hESC-CMs in collagen-based biomaterials and demonstrated that supplementation of mesenchymal niche cells as well as provision of mechanical loading particularly stretching have significantly promoted the maturation of the cardiomyocytes and hence improved the mechanical functional characteristics of the tissue strips. Specifically, with 3% niche cells including both fibroblasts and mesenchymal stem cells, a more mature hESC-CMs derived cardiac strip was resulted, in terms of compaction and spreading of cells, and upregulation of molecular signature in both gene and protein expression of maturation. Mechanical loading, particularly cyclic stretch, produces engineered cardiac tissues with higher maturity in terms of molecular signature markers and functional parameters including twitch force, elastic modulus and sarcomere length, when comparing with static stretch or non-stretched controls. The current study demonstrates that the application of niche cells and mechanical stretch both stimulate the maturation of hESC-CMs in 3D architecture, resulting in more mature cardiac strips. Our results contribute to bioengineering of functional heart tissue strips for drug screening and disease modeling.

Nitric Oxide-cGMP-PKG Pathway Acts on Orai1 to Inhibit the Hypertrophy of Human Embryonic Stem Cell-Derived Cardiomyocytes.

Cardiac hypertrophy is an abnormal enlargement of heart muscle. It frequently results in congestive heart failure, which is a leading cause of human death. Previous studies demonstrated that the nitric oxide (NO), cyclic GMP (cGMP), and protein kinase G (PKG) signaling pathway can inhibit cardiac hypertrophy and thus improve cardiac function. However, the underlying mechanisms are not fully understood. Here, based on the human embryonic stem cell-derived cardiomyocyte (hESC-CM) model system, we showed that Orai1, the pore-forming subunit of store-operated Ca(2+) entry (SOCE), is the downstream effector of PKG. Treatment of hESC-CMs with an α-adrenoceptor agonist phenylephrine (PE) caused a marked hypertrophy, which was accompanied by an upregulation of Orai1. Moreover, suppression of Orai1 expression/activity using Orai1-siRNAs or a dominant-negative construct Orai1(G98A) inhibited the hypertrophy, suggesting that Orai1-mediated SOCE is indispensable for the PE-induced hypertrophy of hESC-CMs. In addition, the hypertrophy was inhibited by NO and cGMP via activating PKG. Importantly, substitution of Ala for Ser(34) in Orai1 abolished the antihypertrophic effects of NO, cGMP, and PKG. Furthermore, PKG could directly phosphorylate Orai1 at Ser(34) and thus prevent Orai1-mediated SOCE. Together, we conclude that NO, cGMP, and PKG inhibit the hypertrophy of hESC-CMs via PKG-mediated phosphorylation on Orai1-Ser-34. These results provide novel mechanistic insights into the action of cGMP-PKG-related antihypertrophic agents, such as NO donors and sildenafil.

Gene- and cell-based bio-artificial pacemaker: what basic and translational lessons have we learned?

Normal rhythms originate in the sino-atrial node, a specialized cardiac tissue consisting of only a few thousands of nodal pacemaker cells. Malfunction of pacemaker cells due to diseases or aging leads to rhythm generation disorders (for example, bradycardias and sick-sinus syndrome (SSS)), which often necessitate the implantation of electronic pacemakers. Although effective, electronic devices are associated with such shortcomings as limited battery life, permanent implantation of leads, lead dislodging, the lack of autonomic responses and so on. Here, various gene- and cell-based approaches, with a particular emphasis placed on the use of pluripotent stem cells and the hyperpolarization-activated cyclic nucleotide-gated-encoded pacemaker gene family, that have been pursued in the past decade to reconstruct bio-artificial pacemakers as alternatives will be discussed in relation to the basic biological insights and translational regenerative potential.

New mechanism of oral immunity to mucosal candidiasis in hyper-IgE syndrome.

Oropharyngeal candidiasis (OPC, thrush) is an opportunistic infection caused by the commensal fungus Candida albicans. An understanding of immunity to Candida has recently begun to unfold with the identification of fungal pattern-recognition receptors such as C-type lectin receptors, which trigger protective T-helper (Th)17 responses in the mucosa. Hyper-IgE syndrome (HIES/Job's syndrome) is a rare congenital immunodeficiency characterized by dominant-negative mutations in signal transducer and activator of transcription 3, which is downstream of the Th17-inductive cytokines interleukin (IL)-6 and IL-23, and hence patients with HIES exhibit dramatic Th17 deficits. HIES patients develop oral and mucocutaneous candidiasis, supporting a protective role for Th17 cells in immunity to OPC. However, the Th17-dependent mechanisms of antifungal immunity in OPC are still poorly defined. An often unappreciated aspect of oral immunity is saliva, which is rich in antimicrobial proteins (AMPs) and exerts direct antifungal activity. In this study, we show that HIES patients show significant impairment in salivary AMPs, including ß-defensin 2 and Histatins. This tightly correlates with reduced candidacidal activity of saliva and concomitantly elevated colonization with Candida. Moreover, IL-17 induces histatins in cultured salivary gland cells. This is the first demonstration that HIES is associated with defective salivary activity, and provides a mechanism for the severe susceptibility of these patients to OPC.

[Cell technologies in complex treatment of venous trophic ulcers].

Live skin equivalent and fibroblasts in gel were used in complex treatment of venous trophic ulcers to evaluate efficacy of cell transplants. Their efficacy depended on extent of trophic ulcer and time of their existence. Cell culture method is minimally traumatic, can be used in elder patients and seniors and gives positive results in 85% of cases.

HCN-encoded pacemaker channels: from physiology and biophysics to bioengineering.

The depolarizing membrane ionic current I(h) (also known as I(f), "f" for funny), encoded by the hyperpolarization-activated cyclic-nucleotide-modulated (HCN1-4) channel gene family, was first discovered in the heart over 25 years ago. Later, I(h) was also found in neurons, retina, and taste buds. HCN channels structurally resemble voltage-gated K(+) (Kv) channels but the molecular features underlying their opposite gating behaviors (activation by hyperpolarization rather than depolarization) and non-selective permeation profiles (> or =25 times less selective for K(+) than Kv channels) remain largely unknown. Although I(h) has been functionally linked to biological processes from the autonomous beating of the heart to pain transmission, the underlying mechanistic actions remain largely inferential and, indeed, somewhat controversial due to the slow kinetics and negative operating voltage range relative to those of the bioelectrical events involved (e.g., cardiac pacing). This article reviews the current state of our knowledge in the structure-function properties of HCN channels in the context of their physiological functions and potential HCN-based therapies via bioengineering.

Molecular architecture of the voltage-dependent Na channel: functional evidence for alpha helices in the pore.

The permeation pathway of the Na channel is formed by asymmetric loops (P segments) contributed by each of the four domains of the protein. In contrast to the analogous region of K channels, previously we (Yamagishi, T., M. Janecki, E. Marban, and G. Tomaselli. 1997. Biophys. J. 73:195-204) have shown that the P segments do not span the selectivity region, that is, they are accessible only from the extracellular surface. The portion of the P-segment NH(2)-terminal to the selectivity region is referred to as SS1. To explore further the topology and functional role of the SS1 region, 40 amino acids NH(2)-terminal to the selectivity ring (10 in each of the P segments) of the rat skeletal muscle Na channel were substituted by cysteine and expressed in tsA-201 cells. Selected mutants in each domain could be blocked with high affinity by externally applied Cd(2)+ and were resistant to tetrodotoxin as compared with the wild-type channel. None of the externally applied sulfhydryl-specific methanethiosulfonate reagents modified the current through any of the mutant channels. Both R395C and R750C altered ionic selectivity, producing significant increases in K(+) and NH(4)(+) currents. The pattern of side chain accessibility is consistent with a pore helix like that observed in the crystal structure of the bacterial K channel, KcsA. Structure prediction of the Na channel using the program PHDhtm suggests an alpha helix in the SS1 region of each domain channel. We conclude that each of the P segments undergoes a hairpin turn in the permeation pathway, such that amino acids on both sides of the putative selectivity filter line the outer mouth of the pore. Evolutionary conservation of the pore helix motif from bacterial K channels to mammalian Na channels identifies this structure as a critical feature in the architecture of ion selective pores.

Latent specificity of molecular recognition in sodium channels engineered to discriminate between two "indistinguishable" mu-conotoxins.

mu-Conotoxins (mu-CTX) are potent oligopeptide blockers of sodium channels. The best characterized forms of mu-CTX, GIIIA and GIIIB, have similar primary and three-dimensional structures and comparable potencies (IC(50) approximately 30 nM) for block of wild-type skeletal muscle Na(+) channels. The two toxins are thus considered to be indistinguishable by their target channels. We have found mutations in the domain II pore region (D762K and E765K) that decrease GIIIB blocking affinity approximately 200-fold, but reduce GIIIA affinity by only approximately 4-fold, compared with wild-type channels. Synthetic mu-CTX GIIIA mutants reveal that the critical residue for differential recognition is at position 14, the site of the only charge difference between the two toxin isoforms. Therefore, engineered Na(+) channels, but not wild-type channels, can discriminate between two highly homologous conotoxins. Latent specificity of toxin-channel interactions, such as that revealed here, is a principle worthy of exploitation in the design and construction of improved biosensors.

Functional consequences of the arrhythmogenic G306R KvLQT1 K+ channel mutant probed by viral gene transfer in cardiomyocytes.

IKs, the slow component of the delayed rectifier potassium current, figures prominently in the repolarization of heart cells. The K+ channel gene KvLQT1 is mutated in the heritable long QT (LQT) syndrome. Heterologous coexpression of KvLQT1 and the accessory protein minK yields an IKs-like current. Nevertheless, the links between KvLQT1 and cardiac IKs are largely inferential. Since the LQT syndrome mutant KvLQT1-G306R suppresses channel activity when coexpressed with wild-type KvLQT1 in a heterologous system, overexpression of this mutant in cardiomyocytes should reduce or eliminate native IKs if KvLQT1 is indeed the major molecular component of this current. To test this idea, we created the adenovirus AdRMGI-KvLQT1-G306R, which overexpresses KvLQT1-G306R channels. In > 60 % of neonatal mouse myocytes, a sizable IKs could be measured using perforated-patch recordings (8.0 +/- 1.6 pA pF-1, n = 13). IKs was increased by forskolin and blocked by clofilium or indapamide but not by E-4031. While cells infected with a reporter virus expressing only green fluorescent protein (GFP) displayed IKs similar to that in uninfected cells, AdRMGI-KvLQT1-G306R-infected cells showed a significantly reduced IKs (2.4 +/- 1.1 pA pF-1, n = 10, P < 0.01) when measured 60-72 h after infection. Similar results were observed in adult guinea-pig myocytes (5.9 +/- 1.2 pA pF-1, n = 9, for control vs. 0.1 +/- 0.1 pA pF-1, n = 5, for AdRMGI-KvLQT1-G306R-infected cells). We conclude that KvLQT1 is the major molecular component of IKs. Our results further establish a dominant-negative mechanism for the G306R LQT syndrome mutation.

Functional roles of cardiac and vascular ATP-sensitive potassium channels clarified by Kir6.2-knockout mice.

-ATP-sensitive potassium (K(ATP)) channels were discovered in ventricular cells, but their roles in the heart remain mysterious. K(ATP) channels have also been found in numerous other tissues, including vascular smooth muscle. Two pore-forming subunits, Kir6.1 and Kir6.2, contribute to the diversity of K(ATP) channels. To determine which subunits are operative in the cardiovascular system and their functional roles, we characterized the effects of pharmacological K(+) channel openers (KCOs, ie, pinacidil, P-1075, and diazoxide) in Kir6.2-deficient mice. Sarcolemmal K(ATP) channels could be recorded electrophysiologically in ventricular cells from Kir6.2(+/+) (wild-type [WT]) but not from Kir6.2(-/-) (knockout [KO]) mice. In WT ventricular cells, pinacidil induced an outward current and action potential shortening, effects that were blocked by glibenclamide, a K(ATP) channel blocker. KO ventricular cells exhibited no response to KCOs, but gene transfer of Kir6.2 into neonatal ventricular cells rescued the electrophysiological response to P-1075. In terms of contractile function, pinacidil decreased force generation in WT but not KO hearts. Pinacidil and diazoxide produced concentration-dependent relaxation in both WT and KO aortas precontracted with norepinephrine. In addition, pinacidil induced a glibenclamide-sensitive current of similar magnitude in WT and KO aortic smooth muscle cells and comparable levels of hypotension in anesthetized WT and KO mice. In both WT and KO aortas, only Kir6.1 mRNA was expressed. These findings indicate that the Kir6.2 subunit mediates the depression of cardiac excitability and contractility induced by KCOs; in contrast, Kir6.2 plays no discernible role in the arterial tree.

Clockwise domain arrangement of the sodium channel revealed by (mu)-conotoxin (GIIIA) docking orientation.

mu-Conotoxins (mu-CTXs) specifically inhibit Na(+) flux by occluding the pore of voltage-gated Na(+) channels. Although the three-dimensional structures of mu-CTXs are well defined, the molecular configuration of the channel receptor is much less certain; even the fundamental question of whether the four homologous Na(+) channel domains are arranged in a clockwise or counter-clockwise configuration remains unanswered. Residues Asp(762) and Glu(765) from domain II and Asp(1241) from domain III of rat skeletal muscle Na(+) channels are known to be critical for mu-CTX binding. We probed toxin-channel interactions by determining the potency of block of wild-type, D762K, E765K, and D1241C channels by wild-type and point-mutated mu-CTXs (R1A, Q14D, K11A, K16A, and R19A). Individual interaction energies for different toxin-channel pairs were quantified from the half-blocking concentrations using mutant cycle analysis. We find that Asp(762) and Glu(765) interact strongly with Gln(14) and Arg(19) but not Arg(1) and that Asp(1241) is tightly coupled to Lys(16) but not Arg(1) or Lys(11). These newly identified toxin-channel interactions within adjacent domains, interpreted in light of the known asymmetric toxin structure, fix the orientation of the toxin with respect to the channel and reveal that the four internal domains of Na(+) channels are arranged in a clockwise configuration as viewed from the extracellular surface.

Molecular basis of electrocardiographic ST-segment elevation.

ST elevation is a classical hallmark of acute transmural myocardial ischemia. Indeed, ST elevation is the major clinical criterion for committing patients with chest pain to emergent coronary revascularization. Despite its clinical importance, the mechanism of ST elevation remains unclear. Various studies have suggested that activation of sarcolemmal ATP-sensitive potassium (K(ATP)) channels by ischemic ATP depletion may play a role, but little direct evidence is available. We studied mice with homozygous knockout (KO) of the Kir6.2 gene, which encodes the pore-forming subunit of cardiac surface K(ATP) channels. Patch-clamp studies in cardiomyocytes confirmed that surface K(ATP) current was indeed absent in KO, but robust in cells from wild-type mice (WT). We then measured continuous electrocardiograms in anesthetized adult mice before and after open-chest ligation of the left anterior descending artery (LAD). Whereas ST elevation was readily evident in WT after LAD ligation, it was markedly suppressed in KO. Such qualitative differences persisted for the rest of the 60-minute observation period of ischemia. In support of the concept that K(ATP) channels are responsible for ST elevation, the surface K(ATP)channel blocker HMR1098 (5 mg/kg IP) suppressed early ST elevation in WT. Thus, the opening of sarcolemmal K(ATP)channels underlies ST elevation during ischemia. These data are the first to link a specific gene product with a common electrocardiographic phenomenon.

Novel structural determinants of mu-conotoxin (GIIIB) block in rat skeletal muscle (mu1) Na+ channels.

mu-Conotoxin (mu-CTX) specifically occludes the pore of voltage-dependent Na(+) channels. In the rat skeletal muscle Na(+) channel (mu1), we examined the contribution of charged residues between the P loops and S6 in all four domains to mu-CTX block. Conversion of the negatively charged domain II (DII) residues Asp-762 and Glu-765 to cysteine increased the IC(50) for mu-CTX block by approximately 100-fold (wild-type = 22.3 +/- 7.0 nm; D762C = 2558 +/- 250 nm; E765C = 2020 +/- 379 nm). Restoration or reversal of charge by external modification of the cysteine-substituted channels with methanethiosulfonate reagents (methanethiosulfonate ethylsulfonate (MTSES) and methanethiosulfonate ethylammonium (MTSEA)) did not affect mu-CTX block (D762C: IC(50, MTSEA+) = 2165.1 +/- 250 nm; IC(50, MTSES-) = 2753.5 +/- 456.9 nm; E765C: IC(50, MTSEA+) = 2200.1 +/- 550.3 nm; IC(50, MTSES-) = 3248.1 +/- 2011.9 nm) compared with their unmodified counterparts. In contrast, the charge-conserving mutations D762E (IC(50) = 21.9 +/- 4.3 nm) and E765D (IC(50) = 22.0 +/- 7.0 nm) preserved wild-type blocking behavior, whereas the charge reversal mutants D762K (IC(50) = 4139.9 +/- 687.9 nm) and E765K (IC(50) = 4202.7 +/- 1088.0 nm) destabilized mu-CTX block even further, suggesting a prominent electrostatic component of the interactions between these DII residues and mu-CTX. Kinetic analysis of mu-CTX block reveals that the changes in toxin sensitivity are largely due to accelerated toxin dissociation (k(off)) rates with little changes in association (k(on)) rates. We conclude that the acidic residues at positions 762 and 765 are key determinants of mu-CTX block, primarily by virtue of their negative charge. The inability of the bulky MTSES or MTSEA side chain to modify mu-CTX sensitivity places steric constraints on the sites of toxin interaction.

Microvascular anastomoses performed in rats using a microsurgical telemanipulator.

OBJECTIVE: To determine the feasibility of performing microsurgical procedures with a remote telemanipulator using a rat femoral artery anastomosis model. MATERIALS AND METHODS: A remote telemanipulator system was developed that enabled precision movements to be performed at up to 30x magnification. Ten 1-mm femoral artery anastomoses were performed in rats using the telemanipulator, and results were compared to those from a control group in which the procedure was performed with conventional microsurgical techniques. Study endpoints included anastomosis completion time, short-term patency, and procedural complications. Statistical analysis was performed using Student's t-test. RESULTS: All anastomoses performed by remote telemanipulation and by conventional microsurgery were completed successfully. Anastomosis completion times were 100.0 +/- 18.6 minutes in the telemanipulator group and 38.8 +/- 5.0 minutes using conventional techniques (p < 0.001). Patency in both groups at 5 minutes and at one hour was 100%. No intraoperative complications were encountered. Postmortem ex vivo examination of the excised arterial segment revealed no technical defects in either group. CONCLUSIONS: Complex procedures requiring a high degree of precision and dexterity can be performed using an electromechanical interface specifically designed for micromanipulation. Performance limitations are similar to those previously reported for remote surgical teleoperation, and most likely reflect incompletely characterized restrictions on multi-sensory information.

Charged residues between the selectivity filter and S6 segments contribute to the permeation phenotype of the sodium channel.

The deep regions of the Na(+) channel pore around the selectivity filter have been studied extensively; however, little is known about the adjacent linkers between the P loops and S6. The presence of conserved charged residues, including five in a row in domain III (D-III), hints that these linkers may play a role in permeation. To characterize the structural topology and function of these linkers, we neutralized the charged residues (from position 411 in D-I and its homologues in D-II, -III, and -IV to the putative start sites of S6) individually by cysteine substitution. Several cysteine mutants displayed enhanced sensitivities to Cd(2+) block relative to wild-type and/or were modifiable by external sulfhydryl-specific methanethiosulfonate reagents when expressed in TSA-201 cells, indicating that these amino acids reside in the permeation pathway. While neutralization of positive charges did not alter single-channel conductance, negative charge neutralizations generally reduced conductance, suggesting that such charges facilitate ion permeation. The electrical distances for Cd(2+) binding to these residues reveal a secondary "dip" into the membrane field of the linkers in domains II and IV. Our findings demonstrate significant functional roles and surprising structural features of these previously unexplored external charged residues.

Local anesthetic anchoring to cardiac sodium channels. Implications into tissue-selective drug targeting.

Local anesthetics inhibit Na+ channels in a variety of tissues, leading to potentially serious side effects when used clinically. We have created a series of novel local anesthetics by connecting benzocaine (BZ) to the sulfhydryl-reactive group methanethiosulfonate (MTS) via variable-length polyethylether linkers (L) (MTS-LX-BZ [X represents 0, 3, 6, or 9]). The application of MTS-LX-BZ agents modified native rat cardiac as well as heterologously expressed human heart (hH1) and rat skeletal muscle (rSkM1) Na+ channels in a manner resembling that of free BZ. Like BZ, the effects of MTS-LX-BZ on rSkM1 channels were completely reversible. In contrast, MTS-LX-BZ modification of heart and mutant rSkM1 channels, containing a pore cysteine at the equivalent location as cardiac Na+ channels (ie, Y401C), persisted after drug washout unless treated with DTT, which suggests anchoring to the pore via a disulfide bond. Anchored MTS-LX-BZ competitively reduced the affinity of cardiac Na+ channels for lidocaine but had minimal effects on mutant channels with disrupted local anesthetic modification properties. These results establish that anchored MTS-LX-BZ compounds interact with the local anesthetic binding site (LABS). Variation in the linker length altered the potency of channel modification by the anchored drugs, thus providing information on the spatial relationship between the anchoring site and the LABS. Our observations demonstrate that local anesthetics can be anchored to the extracellular pore cysteine in cardiac Na+ channels and dynamically interact with the intracellular LABS. These results suggest that nonselective agents, such as local anesthetics, might be made more selective by linking these agents to target-specific anchors.

Pore residues critical for mu-CTX binding to rat skeletal muscle Na+ channels revealed by cysteine mutagenesis.

We have studied mu-conotoxin (mu-CTX) block of rat skeletal muscle sodium channel (rSkM1) currents in which single amino acids within the pore (P-loop) were substituted with cysteine. Among 17 cysteine mutants expressed in Xenopus oocytes, 7 showed significant alterations in sensitivity to mu-CTX compared to wild-type rSkM1 channel (IC50 = 17.5 +/- 2.8 nM). E758C and D1241C were less sensitive to mu-CTX block (IC50 = 220 +/- 39 nM and 112 +/- 24 nM, respectively), whereas the tryptophan mutants W402C, W1239C, and W1531C showed enhanced mu-CTX sensitivity (IC50 = 1.9 +/- 0.1, 4.9 +/- 0.9, and 5.5 +/- 0.4 nM, respectively). D400C and Y401C also showed statistically significant yet modest (approximately twofold) changes in sensitivity to mu-CTX block compared to WT (p < 0.05). Application of the negatively charged, sulfhydryl-reactive compound methanethiosulfonate-ethylsulfonate (MTSES) enhanced the toxin sensitivity of D1241C (IC50 = 46.3 +/- 12 nM) while having little effect on E758C mutant channels (IC50 = 199.8 +/- 21.8 nM). On the other hand, the positively charged methanethiosulfonate-ethylammonium (MTSEA) completely abolished the mu-CTX sensitivity of E758C (IC50 > 1 microM) and increased the IC50 of D1241C by about threefold. Applications of MTSEA, MTSES, and the neutral MTSBN (benzyl methanethiosulfonate) to the tryptophan-to-cysteine mutants partially or fully restored the wild-type mu-CTX sensitivity, suggesting that the bulkiness of the tryptophan's indole group is a determinant of toxin binding. In support of this suggestion, the blocking IC50 of W1531A (7.5 +/- 1.3 nM) was similar to W1531C, whereas W1531Y showed reduced toxin sensitivity (14.6 +/- 3.5 nM) similar to that of the wild-type channel. Our results demonstrate that charge at positions 758 and 1241 are important for mu-CTX toxin binding and further suggest that the tryptophan residues within the pore in domains I, III, and IV negatively influence toxin-channel interaction.

P-loop flexibility in Na+ channel pores revealed by single- and double-cysteine replacements.

Replacement of individual P-loop residues with cysteines in rat skeletal muscle Na+ channels (SkM1) caused an increased sensitivity to current blockade by Cd2+ thus allowing detection of residues lining the pore. Simultaneous replacement of two residues in distinct P-loops created channels with enhanced and reduced sensitivity to Cd2+ block relative to the individual single mutants, suggesting coordinated Cd2+ binding and cross-linking by the inserted sulfhydryl pairs. Double-mutant channels with reduced sensitivity to Cd2+ block showed enhanced sensitivity after the application of sulfhydryl reducing agents. These results allow identification of residue pairs capable of approaching one another to within less than 3.5 A. We often observed that multiple consecutive adjacent residues in one P-loop could coordinately bind Cd2+ with a single residue in another P-loop. These results suggest that, on the time-scale of Cd2+ binding to mutant Na+ channels, P-loops show a high degree of flexibility.

Altered ionic selectivity of the sodium channel revealed by cysteine mutations within the pore.

To explore the role of pore-lining amino acids in Na+ channel ion-selectivity, pore residues were replaced serially with cysteine in cloned rat skeletal muscle Na+ channels. Ionic selectivity was determined by measuring permeability and ionic current ratios of whole-cell currents in Xenopus oocytes. The rSkM1 channels displayed an ionic selectivity sequence Na+ > Li+ > NH4+ > > Cs+ and were impermeable to divalent cations. Replacement of residues in domain IV showed significantly enhanced current and permeability ratios of NH4+ and K+, and negative shifts in the reversal potentials recorded in the presence of external Na+ solutions when compared to cysteine mutants in domains I, II, and III (except K1237C). Mutants in domain IV showed altered selectivity sequences: W1531C (NH4+ > K+ > Na+ > or = Li+ approximately Cs+), D1532C, and G1533C (Na+ > Li+ > or = NH4+ > K+ > Cs+). Conservative replacement of the aromatic residue in domain IV (W1531) with phenylalanine or tyrosine retained Na+ selectivity of the channel while the alanine mutant (W1531A) reduced ion selectivity. A single mutation within the third pore forming region (K1237C) dramatically altered the selectivity sequence of the rSkM1 channel (NH4+ > K+ > Na+ > or = Li+ approximately Cs+) and was permeable to divalent cations having the selectivity sequence Ca2+ > or = Sr2+ > Mg2+ > Ba2+. Sulfhydryl modification of K1237C, W1531C or D1532C with methanethiosulfonate derivatives that introduce a positively charged ammonium group, large trimethylammonium moiety, or a negatively charged sulfonate group within the pore was ineffective in restoring Na+ selectivity to these channels. Selectivity of D1532C mutants could be largely restored by increasing extracellular pH suggesting altering the ionized state at this position influences selectivity. These data suggest that K1237 in domain III and W1531, D1532, and G1533 in domain IV play a critical role in determining the ionic selectivity of the Na+ channel.

The human antiporcine cellular repertoire. In vitro studies of acquired and innate cellular responsiveness.

Discordant xenogeneic transplantation offers a potentially unlimited source of donor organs from easily bred, nonendangered, physiologically compatible animals, but has been limited by the inevitable occurrence of hyperacute rejection (HAR). The potential existence of cell-mediated discordant graft rejection has remained obscured by HAR, and hence is incompletely understood. To define the cellular elements capable of recognition of and subsequent response against discordant tissue in a clinically applicable species combination, we have studied the in vitro interaction of human peripheral blood lymphocytes against 3 porcine B lymphoblastoid cell lines and 6 primary porcine endothelial cell populations. PBL from all individuals tested (n = 10) proliferated in response to culture for 72 hr in xenogeneic mixed lymphocyte culture (XMLC) with cell lines expressing porcine MHC (SLA) class II antigens, while endothelial cultures lacking SLA class II generally failed to evoke a response. The proliferative response to class II-positive cells was attenuated by addition of anti-SLA class II antibody but not by anti-SLA class I antibody. Two endothelial populations expressing class II stimulated an inhibitable proliferative response. The magnitude of the short-term proliferative xenogeneic response was similar to that evoked by fully mismatched allogeneic human B lymphoblastoid stimulators. Additionally, extended XMLC was performed with PBL from 3 individuals. All populations responded with continued proliferation when repeatedly stimulated by porcine cells. This was characterized not only by T cell growth, but by prominent NK cell growth as well. Elucidation of the TCR V beta chain usage patterns by semiquantitative PCR documented selection of TCR transcripts from gene family V beta 2 in each group, complemented by a heterogeneous mixture of other transcripts including V beta 17.1, 20.1, and 6.1, suggesting that direct human TCR binding of porcine cells occurs, and that it is likely to be an individualistic response complemented by a more homogeneous NK response. A 51Cr release assay was utilized to demonstrate that unprimed PBL could also lyse porcine target cells. This cytotoxic response was maintained despite the complete removal of T cells, suggesting that porcine-directed NK cell activity is present prior to the maturation of any T cell response. Cytolysis was also demonstrated in serum-free medium and thus was not mediated solely by antibody-dependent cellular cytotoxicity. Chinese hamster ovary cells transfected with the human T cell receptor accessory molecule CD4 were used to study the ability of this molecule to stabilize the interaction between the human TCR and SLA class II.(ABSTRACT TRUNCATED AT 400 WORDS)