Gain-of-function mutation S422L in the KCNJ8-encoded cardiac K(ATP) channel Kir6.1 as a pathogenic substrate for J-wave syndromes.

BACKGROUND J-wave syndromes have emerged conceptually to encompass the pleiotropic expression of J-point abnormalities including Brugada syndrome (BrS) and early repolarization syndrome (ERS). KCNJ8, which encodes the cardiac K(ATP) Kir6.1 channel, recently has been implicated in ERS following identification of the functionally uncharacterized missense mutation S422L. OBJECTIVE The purpose of this study was to further explore KCNJ8 as a novel susceptibility gene for J-wave syndromes. METHODS Using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing, comprehensive open reading frame/splice site mutational analysis of KCNJ8 was performed in 101 unrelated patients with J-wave syndromes, including 87 with BrS and 14 with ERS. Six hundred healthy individuals were examined to assess the allelic frequency for all variants detected. KCNJ8 mutation(s) was engineered by site-directed mutagenesis and coexpressed heterologously with SUR2A in COS-1 cells. Ion currents were recorded using whole-cell configuration of the patch-clamp technique. RESULTS One BrS case and one ERS case hosted the identical missense mutation S422L, which was reported previously. KCNJ8-S422L involves a highly conserved residue and was absent in 1,200 reference alleles. Both cases were negative for mutations in all known BrS and ERS susceptibility genes. K(ATP) current of the Kir6.1-S422L mutation was increased significantly over the voltage range from 0 to 40 mV compared to Kir6.1-WT channels (n = 16-21; P <.05). CONCLUSION These findings further implicate KCNJ8 as a novel J-wave syndrome susceptibility gene and a marked gain of function in the cardiac K(ATP) Kir6.1 channel secondary to KCNJ8-S422L as a novel pathogenic mechanism for the phenotypic expression of both BrS and ERS.

Different dark conformations function in color-sensitive photosignaling by the sensory rhodopsin I-HtrI complex.

The haloarchaeal phototaxis receptor sensory rhodopsin I (SRI) in complex with its transducer HtrI delivers an attractant signal from excitation with an orange photon and a repellent signal from a second near-UV photon excitation. Using a proteoliposome system with purified SRI in complex with its transducer HtrI, we identified by site-directed fluorescence labeling a site (Ser(155)) on SRI that is conformationally active in signal relay to HtrI. Using site-directed spin labeling of Ser(155)Cys with a nitroxide side chain, we detected a change in conformation following one-photon excitation such that the spin probe exhibits a splitting of the outer hyperfine extrema (2A'(zz)) significantly smaller than that of the electron paramagnetic resonance spectrum in the dark state. The dark conformations of five mutant complexes that do not discriminate between orange and near-UV excitation show shifts to lower or higher 2A'(zz) values correlated with the alterations in their motility behavior to one- and two-photon stimuli. These data are interpreted in terms of a model in which the dark complex is populated by two conformers in the wild type, one that inhibits the CheA kinase (A) and the other that activates it (R), shifted in the dark by mutations and shifted in the wild-type SRI-HtrI complex in opposite directions by one-photon and two-photon reactions.

Defective pituitary function and gamete interactions in $\beta$1,4-galactosyltransferase null mice

Despite much attention, the function of oligosaccharide chains of glycoproteins remains largely unknown. Our understanding of oligosaccharide function in vivo has been limited to the use of reagents and targeted mutations that eliminate entire oligosaccharide chains. However, most, if not all biological functions for oligosaccharides have been attributed to specific terminal sequences on these oligosaccharides, yet there have been few studies to examine the consequences of modifying terminal oligosaccharide structures in vivo. To address this issue, mice were created bearing a targeted mutation in $\beta$1,4-galactosyltransferase, an enzyme responsible for elaboration of many of the proposed biologically-active carbohydrate epitopes. Most galactosyltransferase-null mice died within the first few weeks after birth and were characterized by stunted growth, thin skin, sparse hair, and dehydration. In addition, the adrenal cortices were poorly stratified and spermatogenesis was delayed. The few surviving adults had puffy skin (myxedema), difficulty delivering pups at birth (dystocia), and failed to lactate (agalactosis). All of these defects are consistant with endocrine insufficiency, which was confirmed by markedly decreased levels of serum thyroxine. The anterior pituitary gland appeared functionally delayed in newborn mutant mice, since the constituent cells were quiescent and nonsecretory, unlike that of control littermates. However, the anterior pituitary acquired a normal secretory phenotype during neonatal development, although it remained abnormally small and its glycoprotein hormones were devoid of $\beta$1,4-galactosyl residues. These results support in vitro studies suggesting that incomplete glycosylation of pituitary hormones leads to the creation of hormone antagonists that down regulate subsequent endocrine function producing polyglandular endocrine insufficiency. More surprisingly, the fact that some mice survive this neonatal period indicates the presence of a previously unrecognized compensatory pathway for glycoprotein hormone glycosylation and/or action.^ In addition to its well-studied biosynthetic function in the Golgi complex, a GalTase isoform is also expressed on the sperm surface where it functions as a gamete receptor during fertilization by binding to its oligosaccharide ligand on the egg coat glycoprotein, ZP3. Aggregation of GalTase by multivalent ZP3 oligosaccharides activates a G-protein cascade leading to the acrosome reaction. Although GalTase-null males are fertile, the mutant sperm bind less ZP3 than wild-type sperm, and are unable to undergo the acrosome reaction in response to either zona pellucida glycoproteins or to anti-GalTase anti-serum, as do wild-type sperm. However, mutant and wild-type sperm undergo the acrosome reaction normally in response to calcium ionophore which bypasses the requirement for ZP3 binding. Interestingly, the phenotype of the GalTase-null sperm is reciprocal to that of sperm that overexpress surface GalTAse and which bind more ZP3 leading to precocious acrosome reactions. These results confirm that GalTase functions as at least one of the sperm receptors for ZP3, and that GalTase participates in the ZP3-induced signal transduction pathway during zona pellucida-induced acrosome reactions. ^

PDZ domain-binding motif regulates cardiomyocyte compartment-specific NaV1.5 channel expression and function

BACKGROUND Sodium channel NaV1.5 underlies cardiac excitability and conduction. The last 3 residues of NaV1.5 (Ser-Ile-Val) constitute a PDZ domain-binding motif that interacts with PDZ proteins such as syntrophins and SAP97 at different locations within the cardiomyocyte, thus defining distinct pools of NaV1.5 multiprotein complexes. Here, we explored the in vivo and clinical impact of this motif through characterization of mutant mice and genetic screening of patients. METHODS AND RESULTS To investigate in vivo the regulatory role of this motif, we generated knock-in mice lacking the SIV domain (ΔSIV). ΔSIV mice displayed reduced NaV1.5 expression and sodium current (INa), specifically at the lateral myocyte membrane, whereas NaV1.5 expression and INa at the intercalated disks were unaffected. Optical mapping of ΔSIV hearts revealed that ventricular conduction velocity was preferentially decreased in the transversal direction to myocardial fiber orientation, leading to increased anisotropy of ventricular conduction. Internalization of wild-type and ΔSIV channels was unchanged in HEK293 cells. However, the proteasome inhibitor MG132 rescued ΔSIV INa, suggesting that the SIV motif is important for regulation of NaV1.5 degradation. A missense mutation within the SIV motif (p.V2016M) was identified in a patient with Brugada syndrome. The mutation decreased NaV1.5 cell surface expression and INa when expressed in HEK293 cells. CONCLUSIONS Our results demonstrate the in vivo significance of the PDZ domain-binding motif in the correct expression of NaV1.5 at the lateral cardiomyocyte membrane and underline the functional role of lateral NaV1.5 in ventricular conduction. Furthermore, we reveal a clinical relevance of the SIV motif in cardiac disease.

Glycosylation of TRPM4 and TRPM5 channels: molecular determinants and functional aspects

The transient receptor potential channel, TRPM4, and its closest homolog, TRPM5, are non-selective cation channels that are activated by an increase in intracellular calcium. They are expressed in many cell types, including neurons and myocytes. Although the electrophysiological and pharmacological properties of these two channels have been previously studied, less is known about their regulation, in particular their post-translational modifications. We, and others, have reported that wild-type (WT) TRPM4 channels expressed in HEK293 cells, migrated on SDS-PAGE gel as doublets, similar to other ion channels and membrane proteins. In the present study, we provide evidence that TRPM4 and TRPM5 are each N-linked glycosylated at a unique residue, Asn(992) and Asn(932), respectively. N-linked glycosylated TRPM4 is also found in native cardiac cells. Biochemical experiments using HEK293 cells over-expressing WT TRPM4/5 or N992Q/N932Q mutants demonstrated that the abolishment of N-linked glycosylation did not alter the number of channels at the plasma membrane. In parallel, electrophysiological experiments demonstrated a decrease in the current density of both mutant channels, as compared to their respective controls, either due to the Asn to Gln mutations themselves or abolition of glycosylation. To discriminate between these possibilities, HEK293 cells expressing TRPM4 WT were treated with tunicamycin, an inhibitor of glycosylation. In contrast to N-glycosylation signal abolishment by mutagenesis, tunicamycin treatment led to an increase in the TRPM4-mediated current. Altogether, these results demonstrate that TRPM4 and TRPM5 are both N-linked glycosylated at a unique site and also suggest that TRPM4/5 glycosylation seems not to be involved in channel trafficking, but mainly in their functional regulation.

Effect of large doses of parenteral vitamin D on glycaemic control and calcium/phosphate metabolism in patients with stable type 2 diabetes mellitus: a randomised, placebo-controlled, prospective pilot study.

OBJECTIVE Vitamin D (D₃) status is reported to correlate negatively with insulin production and insulin sensitivity in patients with type 2 diabetes mellitus (T2DM). However, few placebo-controlled intervention data are available. We aimed to assess the effect of large doses of parenteral D3 on glycosylated haemoglobin (HbA(₁c)) and estimates of insulin action (homeostasis model assessment insulin resistance: HOMA-IR) in patients with stable T2DM. MATERIALS AND METHODS We performed a prospective, randomised, double-blind, placebo-controlled pilot study at a single university care setting in Switzerland. Fifty-five patients of both genders with T2DM of more than 10 years were enrolled and randomised to either 300,000 IU D₃ or placebo, intramuscularly. The primary endpoint was the intergroup difference in HbA(₁c) levels. Secondary endpoints were: changes in insulin sensitivity, albuminuria, calcium/phosphate metabolism, activity of the renin-aldosterone axis and changes in 24-hour ambulatory blood pressure values. RESULTS After 6 months of D₃ supply, there was a significant intergroup difference in the change in HbA(₁c) levels (relative change [mean ± standard deviation] +2.9% ± 1.5% in the D₃ group vs +6.9% ± 2.1% the in placebo group, p = 0.041) as HOMA-IR decreased by 12.8% ± 5.6% in the D₃ group and increased by 10% ± 5.4% in the placebo group (intergroup difference, p = 0.032). Twenty-four-hour urinary albumin excretion decreased in the D₃ group from 200 ± 41 to 126 ± 39, p = 0.021). There was no significant intergroup difference for the other secondary endpoints. CONCLUSIONS D₃ improved insulin sensitivity (based on HOMA-IR) and affected the course of HbA(₁c) positively compared with placebo in patients with T2DM.

Dystrophic cardiomyopathy: role of TRPV2 channels in stretch-induced cell damage

Aims Duchenne muscular dystrophy (DMD), a degenerative pathology of skeletal muscle, also induces cardiac failure and arrhythmias due to a mutation leading to the lack of the protein dystrophin. In cardiac cells, the subsarcolemmal localization of dystrophin is thought to protect the membrane from mechanical stress. The absence of dystrophin results in an elevated stress-induced Ca2+ influx due to the inadequate functioning of several proteins, such as stretch-activated channels (SACs). Our aim was to investigate whether transient receptor potential vanilloid channels type 2 (TRPV2) form subunits of the dysregulated SACs in cardiac dystrophy. Methods and results We defined the role of TRPV2 channels in the abnormal Ca2+ influx of cardiomyocytes isolated from dystrophic mdx mice, an established animal model for DMD. In dystrophic cells, western blotting showed that TRPV2 was two-fold overexpressed. While normally localized intracellularly, in myocytes from mdx mice TRPV2 channels were translocated to the sarcolemma and were prominent along the T-tubules, as indicated by immunocytochemistry. Membrane localization was confirmed by biotinylation assays. Furthermore, in mdx myocytes pharmacological modulators suggested an abnormal activity of TRPV2, which has a unique pharmacological profile among TRP channels. Confocal imaging showed that these compounds protected the cells from stress-induced abnormal Ca2+ signals. The involvement of TRPV2 in these signals was confirmed by specific pore-blocking antibodies and by small-interfering RNA ablation of TRPV2. Conclusion Together, these results establish the involvement of TRPV2 in a stretch-activated calcium influx pathway in dystrophic cardiomyopathy, contributing to the defective cellular Ca2+ handling in this disease.

Supramolecular Organization of Dye Molecules in Zeolite L Channels: Synthesis, Properties, and Composite Materials

Sequential insertion of different dyes into the 1D channels of zeolite L (ZL) leads to supramolecular sandwich structures and allows the formation of sophisticated antenna composites for light harvesting, transport, and trapping. The synthesis and properties of dye molecules, host materials, composites, and composites embedded in polymer matrices, including two- and three-color antenna systems, are described. Perylene diimide (PDI) dyes are an important class of chromophores and are of great interest for the synthesis of artificial antenna systems. They are especially well suited to advancing our understanding of the structure–transport relationship in ZL because their core fits tightly through the 12-ring channel opening. The substituents at both ends of the PDIs can be varied to a large extent without influencing their electronic absorption and fluorescence spectra. The intercalation/insertion of 17 PDIs, 2 terrylenes, and 1 quaterrylene into ZL are compared and their interactions with the inner surface of the ZL nanochannels discussed. ZL crystals of about 500 nm in size have been used because they meet the criteria that must be respected for the preparation of antenna composites for light harvesting, transport, and trapping. The photostability of dyes is considerably improved by inserting them into the ZL channels because the guests are protected by being confined. Plugging the channel entrances, so that the guests cannot escape into the environment is a prerequisite for achieving long-term stability of composites embedded in an organic matrix. Successful methods to achieve this goal are described. Finally, the embedding of dye–ZL composites in polymer matrices, while maintaining optical transparency, is reported. These results facilitate the rational design of advanced dye–zeolite composite materials and provide powerful tools for further developing and understanding artificial antenna systems, which are among the most fascinating subjects of current photochemistry and photophysics.

Use of Calcium Channel Blockers is Associated with Mortality in Patients with Chronic Kidney Disease.

BACKGROUND/AIMS The use of antihypertensive medicines has been shown to reduce proteinuria, morbidity, and mortality in patients with chronic kidney disease (CKD). A specific recommendation for a class of antihypertensive drugs is not available in this population, despite the pharmacodynamic differences. We have therefore analysed the association between antihypertensive medicines and survival of patients with chronic kidney disease. METHODS Out of 2687 consecutive patients undergoing kidney biopsy a cohort of 606 subjects with retrievable medical therapy was included into the analysis. Kidney function was assessed by glomerular filtration rate (GFR) estimation at the time point of kidney biopsy. Main outcome variable was death. RESULTS Overall 114 (18.7%) patients died. In univariate regression analysis the use of alpha-blockers and calcium channel antagonists, progression of disease, diabetes mellitus (DM) type 1 and 2, arterial hypertension, coronary heart disease, peripheral vascular disease, male sex and age were associated with mortality (all p<0.05). In a multivariate Cox regression model the use of calcium channel blockers (HR 1.89), age (HR 1.04), DM type 1 (HR 8.43) and DM type 2 (HR 2.17) and chronic obstructive pulmonary disease (HR 1.66) were associated with mortality (all p < 0.05). CONCLUSION The use of calcium channel blockers but not of other antihypertensive medicines is associated with mortality in primarily GN patients with CKD.

Renal function can be impaired in children with primary hyperoxaluria type 3

BACKGROUND Primary hyperoxaluria type 3 (PH3) is characterized by mutations in the 4-hydroxy-2-oxoglutarate aldolase (HOGA1) gene. PH3 patients are believed to present with a less severe phenotype than those with PH1 and PH2, but the clinical characteristics of PH3 patients have yet to be defined in sufficient detail. The aim of this study was to report our experience with PH3. METHODS Genetic analysis of HOGA1 was performed in patients with a high clinical suspicion of PH after the presence of mutations in the alanine-glyoxylate aminotransferase gene had been ruled out. Clinical, biochemical and genetic data of the seven patients identified with HOGA1 mutations were subsequently retrospectively reviewed. RESULTS Among the seven patients identified with HOGA1 mutations the median onset of clinical symptoms was 1.8 (range 0.4-9.8) years. Five patients initially presented with urolithiasis, and two other patients presented with urinary tract infection. All patients experienced persistent hyperoxaluria. Seven mutations were found in HOGA1, including two previously unreported ones, c.834 + 1G > T and c.3G > A. At last follow-up, two patients had impaired renal function based on estimated glomerular filtration rates (GFRs) of 77 and 83 mL/min per 1.73 m(2), respectively. CONCLUSIONS We found that the GFR was significantly impaired in two of our seven patients with PH3 diagnosed during childhood. This finding is in contrast to the early-impaired renal function in PH1 and PH2 and appears to refute to preliminary reassuring data on renal function in PH3.

An extracytoplasmic function sigma factor operon regulates Myxococcus xanthus developmental gene expression

Myxococcus xanthus is a Gram-negative soil bacterium that undergoes multicellular development when high-density cells are starved on a solid surface. Expression of the 4445 gene, predicted to encode a periplasmic protein, commences 1.5 h after the initiation of development and requires starvation and high density conditions. Addition of crude or boiled supernatant from starving high-density cells restored 4445 expression to starving low-density cells. Addition of L-threonine or L-isoleucine to starving low-density cells also restored 4445 expression, indicating that the high-density signaling activity present in the supernatant might be composed of extracellular amino acids or small peptides. To investigate the circuitry integrating these starvation and high-density signals, the cis- and trans-acting elements controlling 4445 expression were identified. The 4445 transcription start site was determined by primer extension analysis to be 58 by upstream of the predicted translation start site. The promoter region contained a consensus sequence characteristic of e&barbelow;xtrac&barbelow;ytoplasmic f&barbelow;unction (ECF) sigma factor-dependent promoters, suggesting that 4445 expression might be regulated by an ECF sigma factor-dependent pathway, which are known to respond to envelope stresses. The small size of the minimum regulatory region, identified by 5′-end deletion analysis as being only 66 by upstream of the transcription start site, suggests that RNA polymerase could be the sole direct regulator of 4445 expression. To identify trans-acting negative regulators of 4445 expression, a strain containing a 4445-lacZ was mutagenized using the Himar1-tet transposon. The four transposon insertions characterized mapped to an operon encoding a putative ECF sigma factor, ecfA; an anti-sigma factor, reaA; and a negative regulator, reaB. The reaA and the reaB mutants expressed 4445 during growth and development at levels almost 100-fold higher than wild type, indicating that these genes encode negative regulators. The ecfA mutant expressed 4445-lacZ at basal levels, indicating that ecfA is a positive regulator. High Mg2+ concentrations over-stimulated this ecfA pathway possibly due to the depletion of exopolysaccharides and assembled type IV pili. These data indicate that the ecfA operon encodes a new regulatory stress pathway that integrates and transduces starvation and cell density cues during early development and is also responsive to cell-surface alterations.^

Role of TRP Channels in Mediating the Calcium Signaling Response of Brain Endothelial Cells to Mechanical Stretch

Traumatic brain injury (TBI) often results in disruption of the blood brain barrier (BBB), which is an integral component to maintaining the central nervous system homeostasis. Recently cytosolic calcium levels ([Ca2+]i), observed to elevate following TBI, have been shown to influence endothelial barrier integrity. However, the mechanism by which TBI-induced calcium signaling alters the endothelial barrier remains unknown. In the present study, an in vitro BBB model was utilized to address this issue. Exposure of cells to biaxial mechanical stretch, in the range expected for TBI, resulted in a rapid cytosolic calcium increase. Modulation of intracellular and extracellular Ca2+ reservoirs indicated that Ca2+ influx is the major contributor for the [Ca2+]i elevation. Application of pharmacological inhibitors was used to identify the calcium-permeable channels involved in the stretch-induced Ca2+ influx. Antagonist of transient receptor potential (TRP) channel subfamilies, TRPC and TRPP, demonstrated a reduction of the stretch-induced Ca2+ influx. RNA silencing directed at individual TRP channel subtypes revealed that TRPC1 and TRPP2 largely mediate the stretch-induced Ca2+ response. In addition, we found that nitric oxide (NO) levels increased as a result of mechanical stretch, and that inhibition of TRPC1 and TRPP2 abolished the elevated NO synthesis. Further, as myosin light chain (MLC) phosphorylation and actin cytoskeleton rearrangement are correlated with endothelial barrier disruption, we investigated the effect mechanical stretch had on the myosin-actin cytoskeleton. We found that phosphorylated MLC was increased significantly by 10 minutes post-stretch, and that inhibition of TRP channel activity or NO synthesis both abolished this effect. In addition, actin stress fibers formation significantly increased 2 minutes post-stretch, and was abolished by treatment with TRP channel inhibitors. These results suggest that, in brain endothelial cells, TRPC1 and TRPP2 are activated by TBI-mechanical stress and initiate actin-myosin contraction, which may lead to disruption of the BBB.

Expression and function of $\beta$-1,4-galactosyltransferase during wild-type and T/{\it t\/} -mutant spermatogenesis

In the mouse, gamete recognition is mediated in part by the binding of sperm surface $\beta$1,4 galactosyltransferase (GalTase) to specific oligosaccharide residues on the zona pellucida ZP3. The expression of GalTase on the sperm surface is regulated by alleles within the distal segment of the T/t complex and results in a haploid-specific increase in GalTase expression on spermatids and sperm from t-bearing males, suggesting that differences in sperm GalTase activity may contribute to t-sperm transmission ratio distortion. In this study, the expression of GalTase RNA during wild-type and T/t-mutant spermatogenesis was characterized and the role of GalTase was analyzed in transmission ratio distortion. It was found that spermatogenic cells predominantly express the long form of the GalTase RNA, which encodes the GalTase protein that is preferentially targeted to the cell surface in somatic cells. In wild-type testes, GalTase RNA accumulates during the maturation of primary spermatocytes, reaches peak levels prior to meiosis, and decreases and meiosis. GalTase RNA accumulates to similar levels during the maturation of +/t and t/t primary spermatocytes, but unlike wild-type, the level of GalTase RNA in t-spermatocytes remains elevated during meiotic division. Consequently, spermatids in t-mutant testes inherit higher levels of GalTase RNA than do wild-type spermatids, which likely accounts for the haploid-specific increase in surface GalTase activity characteristic of spermatids from t-bearing mice.^ The functional significance of the increased GalTase activity during t-sperm transmission ratio distortion was determined by examining the distribution of GalTase RNA and surface GalTase protein in haploid spermatids from +/t males. Results show that +- and t-spermatids have similar levels of both GalTase RNA and protein, indicating that transmission ratio distortion in +/t mice is not likely due to haploid-specific differences in sperm surface GalTase activity.^ The presence of GalTase on the surface of an early spermatogenic cells before it is required on the mature sperm to perform its function during gamete binding suggests a separate function for GalTase in Sertoli-germ cell adhesion. Studies indicate that cell surface GalTase partly mediates the initial adhesion of pachytene spermatocytes, but not haploid spermatids, to Sertoli cells. ^

Evidence that lymphokine -activated killer (LAK) cell function is regulated by both serine and tyrosine kinase pathways

Signal transduction pathways operative in lymphokine activated killer (LAK) cells during execution of cytolytic function have never been characterized. Based on ubiquitous involvement of protein phosphorylation in activation of cytolytic mechanisms used by CTL and NK cells, it was hypothesized that changes in protein phosphorylation should occur when LAK encounter tumor targets. It was further hypothesized that protein kinases would regulate LAK-mediated cytotoxicity. Exposure to either SK-Mel-1 (melanoma) or Raji (lymphoma) targets consistently led to increased phosphorylation of two 65-kD LAK proteins pp65a and -b, with isoelectric points (pI) of 5.1 and 5.2 respectively. Increased p65 phosphorylation was initiated between 1 and 5 min after tumor coincubation, occurred on Ser residues, required physical contact between LAK and tumors, correlated with target recognition, and also occurred after crosslinking Fc$\gamma$RIIIA in the absence of tumors. Both pp65a and -b were tentatively identified as phosphorylated forms of the actin-bundling protein L-plastin, based on pI, molecular weight, and cross-reactivity with specific antiserum. The known biochemical properties of L-plastin suggest it may be involved in regulating adhesion of LAK to tumor targets. The protein tyrosine kinase-specific inhibitor Herb A did not block p65 phosphorylation, but blocked LAK killing of multiple tumor targets at a post-binding stage. Greater than 50% inhibition of cytotoxicity was observed after a 2.5-h pretreatment with 0.125 $\mu$g/ml Herb A. Inhibition occurred over a period in pretreatment which LAK were not dependent upon IL-2 for maintenance of killing activity, supporting the conclusion that the drug interfered with mobilization of cytotoxic function. Granule exocytosis measured by BLT-esterase release from LAK occurred after coincubation with tumors, and was inhibited by Herb A LAK cytotoxicity was dependent upon extracellular calcium, suggesting that granule exocytosis rather than Fas ligand was the principal pathway leading to target cell death. The data indicate that protein tyrosine kinases play a pivotal role in LAK cytolytic function by regulating granule exocytosis, and that tumor targets can activate an adhesion dependent Ser kinase pathway in LAK resulting in phosphorylation of L-plastin. ^