Exome Sequencing of Extended Families with Alzheimer's Disease Identifies Novel Genes Implicated in Cell Immunity and Neuronal Function.

OBJECTIVE: Alzheimer's disease (AD) is a neurodegenerative disorder for which more than 20 genetic loci have been implicated to date. However, studies demonstrate not all genetic factors have been identified. Therefore, in this study we seek to identify additional rare variants and novel genes potentially contributing to AD. METHODS: Whole exome sequencing was performed on 23 multi-generational families with an average of eight affected subjects. Exome sequencing was filtered for rare, nonsynonymous and loss-of-function variants. Alterations predicted to have a functional consequence and located within either a previously reported AD gene, a linkage peak (LOD>2), or clustering in the same gene across multiple families, were prioritized. RESULTS: Rare variants were found in known AD risk genes including AKAP9, CD33, CR1, EPHA1, INPP5D, NME8, PSEN1, SORL1, TREM2 and UNC5C. Three families had five variants of interest in linkage regions with LOD>2. Genes with segregating alterations in these peaks include CD163L1 and CLECL1, two genes that have both been implicated in immunity, CTNNA1, which encodes a catenin in the cerebral cortex and MIEF1, a gene that may induce mitochondrial dysfunction and has the potential to damage neurons. Four genes were identified with alterations in more than one family include PLEKHG5, a gene that causes Charcot-Marie-Tooth disease and THBS2, which promotes synaptogenesis. CONCLUSION: Utilizing large families with a heavy burden of disease allowed for the identification of rare variants co-segregating with disease. Variants were identified in both known AD risk genes and in novel genes.

Areawide suppression of European corn borer with Bt maize reaps savings to non-Bt maize growers.

Transgenic maize engineered to express insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) has become widely adopted in U.S. agriculture. In 2009, Bt maize was planted on more than 22.2 million hectares, constituting 63% of the U.S. crop. Using statistical analysis of per capita growth rate estimates, we found that areawide suppression of the primary pest Ostrinia nubilalis (European corn borer) is associated with Bt maize use. Cumulative benefits over 14 years are an estimated $3.2 billion for maize growers in Illinois, Minnesota, and Wisconsin, with more than $2.4 billion of this total accruing to non-Bt maize growers. Comparable estimates for Iowa and Nebraska are $3.6 billion in total, with $1.9 billion for non-Bt maize growers. These results affirm theoretical predictions of pest population suppression and highlight economic incentives for growers to maintain non-Bt maize refugia for sustainable insect resistance management.

Management of hilar bile duct carcinoma.

BACKGROUND/AIMS: Hilar cholangiocarcinoma is a rare tumor with a dismal prognosis. Because proximal bile duct cancers are uncommon, outcomes related to various therapeutic interventions are not well defined. METHODOLOGY: Between 1985 and 1997, 55 patients with bile duct cancers involving the proximal third of the extrahepatic bile ducts were seen. The management of patients with resectable and unresectable disease was retrospectively reviewed. All but four patients were followed until the time of death. RESULTS: Forty patients underwent laparotomy following preoperative assessment of extent of disease and 19 patients (35%) ultimately underwent resection with curative intent. Survival was significantly longer in patients who underwent resection (2-year survival 47% vs. 18%; P = 0.027). Of those patients whose disease was resected, 11 patients received adjuvant radiotherapy. Survival for this group was not significantly different from that seen in patients who did not receive adjuvant radiotherapy. Similarly, in patients with unresectable disease, administration of radiotherapy was not associated with an improved outcome. CONCLUSIONS: Locoregional extent of disease is the greatest problem in cases of proximal bile duct cancers. Resection provides the best hope for long-term survival, but new adjuvant strategies are needed.

Short-term exercise training can improve myocardial tolerance to I/R without elevation in heat shock proteins.

We examined the effects of 3 days of exercise in a cold environment on the expression of left ventricular (LV) heat shock proteins (HSPs) and contractile performance during in vivo ischemia-reperfusion (I/R). Sprague-Dawley rats were divided into the following three groups (n = 12/group): 1) control, 2) exercise (60 min/day) at 4 degrees C (E-Cold), and 3) exercise (60 min/day) at 25 degrees C (E-Warm). Left anterior descending coronary occlusion was maintained for 20 min, followed by 30 min of reperfusion. Compared with the control group, both the E-Cold and E-Warm groups maintained higher (P < 0.05) LV developed pressure, first derivative of pressure development over time (+dP/dt), and pressure relaxation over time (-dP/dt) throughout I/R. Relative levels of HSP90, HSP72, and HSP40 were higher (P < 0.05) in E-Warm animals compared with both control and E-Cold. HSP10, HSP60, and HSP73 did not differ between groups. Exercise increased manganese superoxide dismutase (MnSOD) activity in both E-Warm and E-Cold hearts (P < 0.05). Protection against I/R-induced lipid peroxidation in the LV paralleled the increase in MnSOD activity whereas lower levels of lipid peroxidation were observed in both E-Warm and E-Cold groups compared with control. We conclude that exercise-induced myocardial protection against a moderate duration I/R insult is not dependent on increases in myocardial HSPs. We postulate that exercise-associated cardioprotection may depend, in part, on increases in myocardial antioxidant defenses.

Diminished benefit from resection of cancer of the head of the pancreas in patients of advanced age.

BACKGROUND AND OBJECTIVES: The incidence of pancreatic cancer is increasing, and an increasing proportion of these patients is older than 65 years. The benefits of resection in the geriatric population, in whom major comorbidity is more likely, are poorly defined. The authors sought to determine the relative benefits of resection of cancer of the head of the pancreas in different age groups, with particular emphasis on the geriatric population. METHODS: Between 1983 and 1995, 273 patients presented to the University of Miami for evaluation of noncystic epithelial cancer of the head of the pancreas. Resection was performed in 104 patients, and these patients are the subject of this retrospective review. Mean length of follow-up for surviving patients was 37 +/- 24 months. Outcomes were compared in patients < 65 years old (group 1, n = 38), 65-74 years old (group 2, n = 47), and > 74 years old (group 3, n = 19). RESULTS: Total pancreatectomy was performed in 12 patients and pancreaticoduodenectomy was performed in 92 patients. The overall complication rate was similar in all groups, but major morbidity was highest in group 3 (P = 0.05). Median survival for patients in group 2 was 25.1 months. Survival was significantly shorter in patients from groups 1 and 3 (median survivals 12.4 months and 11.4 months, respectively; P = 0.02). Following control for Hispanic ethnicity, which was also a significant prognostic factor on univariate analysis, only the oldest age group had a significantly shorter survival than the other two groups. Age > 74 years and Hispanic ethnicity remained significant after multivariate analysis. CONCLUSIONS: Long-term survival after resection is truncated in older patients. This finding and the observation that the major complication rate is higher in the older subgroup emphasize the need to evaluate critically whether older patients should be submitted to radical resection.

HCO3- potentiates the cAMP-dependent secretory response of the human distal colon through a DIDS-sensitive pathway.

We used the Ussing short-circuit technique to investigate the role of HCO3- in the adenosine 3',5'-cyclic monophosphate (cAMP)-dependent secretory response of the human distal colon. In HCO3(-)-free 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES)-Ringer's, forskolin (10 mumol l-1 mucosal and serosal) evoked a sustained increase in short-circuit current (Isc) (delta Isc = 24 +/- 3 microA cm-2, n = 57). However, this was only 25% of the forskolin-stimulated Isc response in HCO3(-)-Ringer's (delta Isc = 84 +/- 8 microA cm-2, n = 57). The reduced response to forskolin in HCO3(-)-free HEPES-Ringer's was not due to inhibition of the secretory mechanism by HEPES, as replacing HCO3- with a different buffer, N-tris[hydroxymethyl)methyl-2-aminoethanesulphonic acid (TES), had a similar effect and inclusion of HEPES in the HCO3(-)-Ringer's did not reduce the secretory response. Similarly, it was not due to an indirect modulation of electrogenic Cl- secretion, as the forskolin-stimulated bumetanide-sensitive Isc was comparable in the two Ringer's. Rather it was due to the activation of a HCO3(-)-dependent Isc which was inhibited by serosal 4,4'-diisothiocyano-stilbene-2,2'-disulphonate (DIDS). This DIDS-sensitive Isc was not inhibited by acetazolamide, but it was inhibited by the replacement of bathing solution Cl- with gluconate, suggesting a role for a Na(+)-dependent Cl-/HCO3- exchanger in the cAMP-dependent secretory response of the human distal colon.

Effects of vitamin E and alpha-lipoic acid on skeletal muscle contractile properties.

Initial experiments were conducted using an in situ rat tibialis anterior (TA) muscle preparation to assess the influence of dietary antioxidants on muscle contractile properties. Adult Sprague-Dawley rats were divided into two dietary groups: 1) control diet (Con) and 2) supplemented with vitamin E (VE) and alpha-lipoic acid (alpha-LA) (Antiox). Antiox rats were fed the Con rats' diet (AIN-93M) with an additional 10,000 IU VE/kg diet and 1.65 g/kg alpha-LA. After an 8-wk feeding period, no differences existed (P > 0.05) between the two dietary groups in maximum specific tension before or after a fatigue protocol or in force production during the fatigue protocol. However, in unfatigued muscle, maximal twitch tension and tetanic force production at stimulation frequencies < or = 40 Hz were less (P < 0.05) in Antiox animals compared with Con. To investigate which antioxidant was responsible for the depressed force production, a second experiment was conducted using an in vitro rat diaphragm preparation. Varying concentrations of VE and dihydrolipoic acid, the reduced form of alpha-LA, were added either individually or in combination to baths containing diaphragm muscle strips. The results from these experiments indicate that high levels of VE depress skeletal muscle force production at low stimulation frequencies.

Improved cardiac performance after ischemia in aged rats supplemented with vitamin E and alpha-lipoic acid.

The purpose of these experiments was to examine the effects of dietary antioxidant supplementation with vitamin E (VE) and alpha-lipoic acid (alpha-LA) on biochemical and physiological responses to in vivo myocardial ischemia-reperfusion (I-R) in aged rats. Male Fischer-334 rats (18 mo old) were assigned to either 1) a control diet (CON) or 2) a VE and alpha-LA supplemented diet (ANTIOX). After a 14-wk feeding period, animals in each group underwent an in vivo I-R protocol (25 min of myocardial ischemia and 15 min of reperfusion). During reperfusion, peak arterial pressure was significantly higher (P < 0.05) in ANTIOX animals compared with CON diet animals. I-R resulted in a significant increase (P < 0.05) in myocardial lipid peroxidation in CON diet animals but not in ANTIOX animals. Compared with ANTIOX animals, heart homogenates from CON animals experienced significantly less (P < 0.05) oxidative damage when exposed to five different in vitro radical producing systems. These data indicate that dietary supplementation with VE and alpha-LA protects the aged rat heart from I-R-induced lipid peroxidation by scavenging numerous reactive oxygen species. Importantly, this protection is associated with improved cardiac performance during reperfusion.

Oral creatine supplementation and upper extremity anaerobic response in females.

The purpose of this study was to investigate the influence of creatine monohydrate (CrH2O) on upper extremity anaerobic response in strength-trained females involved in overhand sports. Two movements were utilized in this evaluation: elbow flexion (EF) and shoulder internal rotation (IR). Subjects were pair-matched and assigned to receive placebo (n = 13) or 25 g CrH2O (n = 11) for 7 days. Pre- and post-treatment measurements included peak concentric and eccentric isokinetic torque, isotonic 1RM, and fatigue (FAT) during EF; isotonic 1RM, FAT, and peak velocity during IR; and body weight. MANOVAs revealed significant interaction between treatment and trial for EF (p <.05) but not for IR or weight. Univariate analysis indicated a significantly greater change in EFFAT following CrH2O than following placebo. Thus, CrH2O did not influence peak EF or IR strength, IR work to fatigue, or IR velocity, but was associated with greater work capacity during fatiguing EF. These data suggest that CrH2O may enhance upper extremity work capacity, but this enhancement may not extend to the muscles primarily responsible for overhand sports performance.

The molecular basis of renal tubular transport disorders.

Sodium and water homeostasis are key to the survival of organisms. Reabsorption of sodium and water occurs throughout the tubule structure of the nephron, the basic functional unit of the kidney, by various transport mechanisms. Altered transport protein function can lead to renal tubular disorders resulting in metabolic alkalosis, hypokalemia, hypertension, and decreased capacity to concentrate urine, for instance. However, recent advances in molecular physiology, molecular genetics and expression cloning systems have aided in unraveling the molecular basis of some renal tubular disorders. This review will examine the molecular basis of Bartter's syndrome, Gitelman's syndrome, Liddle's syndrome, and autosomal nephrogenic diabetes insipidus. An understanding of the molecular basis of these disorders of the human kidney can give us a better understanding of basic renal function of lower mammals and other vertebrates.

Effect of combined supplementation with vitamin E and alpha-lipoic acid on myocardial performance during in vivo ischaemia-reperfusion.

Reactive oxygen species (ROS) contribute significantly to myocardial ischaemia-reperfusion (I-R) injury. Recently the combination of the antioxidants vitamin E (VE) and alpha-lipoic acid (alpha-LA) has been reported to improve cardiac performance and reduce myocardial lipid peroxidation during in vitro I-R. The purpose of these experiments was to investigate the effects of VE and alpha-LA supplementation on cardiac performance, incidence of dysrhythmias and biochemical alterations during an in vivo myocardial I-R insult. Female Sprague-Dawley rats (4-months old) were assigned to one of the two dietary treatments: (1) control diet (CON) or (2) VE and alpha-LA supplementation (ANTIOXID). The CON diet was prepared to meet AIN-93M standards, which contains 75 IU VE kg-1 diet. The ANTIOXID diet contained 10 000 IU VE kg(-1) diet and 1.65 g alpha-LA kg(-1) diet. After the 14-week feeding period, significant differences (P<0.05) existed in mean myocardial VE levels between dietary groups. Animals in each experimental group were subjected to an in vivo I-R protocol which included 25 min of left anterior coronary artery occlusion followed by 10 min of reperfusion. No group differences (P>0.05) existed in cardiac performance (e.g. peak arterial pressure or ventricular work) or the incidence of ventricular dysrhythmias during the I-R protocol. Following I-R, two markers of lipid peroxidation were lower (P<0.05) in the ANTIOXID animals compared with CON. These data indicate that dietary supplementation of the antioxidants, VE and alpha-LA do not influence cardiac performance or the incidence of dysrhythmias but do decrease lipid peroxidation during in vivo I-R in young adult rats.

Effect of somatostatin on electrogenic ion transport in the duodenum and colon of the mouse, Mus domesticus.

In this study, we have used the mouse intestine and the Ussing short circuit technique to compare the effects and mechanism of action of somatostatin (SST, 0.1 microM) on cAMP- and Ca(2+)-mediated ion secretion in the duodenum and colon of the Swiss-Webster mouse. The cAMP-dependent secretagogues, prostaglandin E(2) (1 microM) and dibutyryl-cAMP (150 microM) increased short circuit current (I(sc)) in both regions, but only the colonic response was inhibited by SST. This inhibition was independent of enteric nerves, suggesting a direct action on the epithelial cells. The Ca(2+)-dependent secretagogue carbachol (10 microM) stimulated a transient increase in I(sc) in both intestinal segments. In the duodenum, SST partially inhibited this increase in I(sc) and both the responses to carbachol and SST were independent of enteric nerves. In the colon, while SST inhibited the carbachol induced increase in I(sc), pre-treatment with tetrodotoxin (750 nM) profoundly inhibited the carbachol induced increase in I(sc), thus markedly reducing the inhibitory effect of SST. This indicates an involvement of the enteric nervous system in the response to carbachol and the action of SST in the colon. These data indicate marked regional differences within the mouse intestine of the effects of SST on ion secretion and demonstrate different mechanisms of action of SST in the duodenum and colon.

The effectiveness of commercially available sports drinks.

The purpose of this review is to evaluate the effectiveness of commercially available sports drinks by answering the questions: (i) will consuming a sports drink be beneficial to performance? and (ii) do different sports drinks vary in their effectiveness? To answer these questions we have considered the composition of commercially available sports drinks, examined the rationale for using them, and critically reviewed the vast number of studies that have investigated the effectiveness of sports drinks on performance. The focus is on the drinks that contain low carbohydrate concentrations (<10%) and are marketed for general consumption before and during exercise rather than those with carbohydrate concentrations >10%, which are intended for carbohydrate loading. Our conclusions are 3-fold. First, because of variations in drink composition and research design, much of the sports drinks research from the past cannot be applied directly to the effectiveness of currently available sports drinks. Secondly, in studies where a practical protocol has been used along with a currently available sports beverage, there is evidence to suggest that consuming a sports drinks will improve performance compared with consuming a placebo beverage. Finally, there is little evidence that any one sports drink is superior to any of the other beverages on the market.

Vitamin E deficiency fails to affect myocardial performance during in vivo ischemia-reperfusion.

Vitamin E content of cardiac tissue has been proposed to play a major role in the damage caused by myocardial ischemia-reperfusion (I-R). Previous studies using in vitro models have examined vitamin E deficiency and I-R-induced myocardial damage with equivocal results. The purpose of this study was to use an in vivo model of myocardial I-R to determine the effects of vitamin E deficiency on myocardial I-R-induced damage. Female Sprague-Dawley rats (4-mo old) were assigned to either: 1) control diet (CON), or 2) vitamin E deficient diet (VE-DEF). The CON diet was prepared to meet AIN-93M standards, which contains 75 IU vitamin E/kg diet. The VE-DEF diet was the AIN-93M diet prepared with tocopherol stripped corn oil and no vitamin E. Following a 14-week feeding period, significant differences (p < 0.05) existed in mean myocardial VE levels between groups (mean values +/- SEM: CON = 48.2 +/- 3.5; VE-DEF = 12.4 +/- 1.4 micrograms VE/g wet weight). Animals from both experimental groups were subjected to an in vivo I-R protocol consisting of 25 minutes of left coronary artery occlusion followed by 10 minutes of reperfusion. No group differences (p > 0.05) existed in cardiac performance (peak arterial pressure or ventricular work) or the incidence of ventricular arrhythmias during the I-R protocol. VE-DEF animals had significantly higher (p < 0.05) levels of myocardial lipid peroxidation and lower (p < 0.05) protein thiols following I-R compared to the CON animals. These data suggest that although vitamin E deficiency increases oxidative damage resulting from myocardial I-R, it does not affect cardiac performance during the insult.

1-EBIO stimulates Cl- secretion by activating a basolateral K+ channel in the mouse jejunum.

We investigated the effects of 1-ethyl-2-benzimidazolinone (1-EBIO) on ion transport in the mouse jejunum through the use of the short-circuit (Isc) current technique and the application of the patch-clamp technique to isolated jejunal crypts. In HCO3- Ringer's, 1-EBIO stimulated a dose-dependent (EC50 964 micromol/l), bumetanide-sensitive increase in Isc consistent with stimulation of Cl- secretion. In contrast, in Cl(-)-free HCO3-Ringer's containing glucose, 1-EBIO (500 micromol/l) did not increase the phloridzin (100 micromol/l) sensitive Isc, suggesting that electrogenic Na+ absorption was unaltered. Measurement of the membrane potential (Vm) with the perforated-patch technique indicated that in isolated crypts, 1-EBIO caused a reversible hyperpolarization of Vm and an increase in the change in Vm associated with step changes in bath K+, consistent with an increase in K+ conductance. In on-cell patch experiments with KCI Ringer's in the patch pipette and crypts bathed with NaCl Ringer's, 1-EBIO (500 micromol/l) increased the open probability (NPo; 0.01+/-0.01 to 0.45+/-0.11, n=7) of an inwardly rectified intermediate conductance (g) channel. In inside-out patches with KCl Ringer's in the patch pipette and KCI Ringer's containing 100 nmol/l Ca2+ in the bath, the current-voltage relationship of the channel was inwardly rectified (g of 10 and 52 pS at -Vp of 100 and -100 mV, respectively) and reversed at 0 mV (n=5). Replacement of bath K+ with Na+ shifted the reversal potential toward the equilibrium potential for K+. In the presence of 1-EBIO, reducing the bath Ca2+ from 200 nmol/l to nominally Ca(2+)-free conditions decreased NPo from 0.90+/-0.27 to 0.07+/-0.03 (n=3). We conclude that in the mouse jejunum, I-EBIO does not stimulate electrogenic Na+ absorption. It does, however, stimulate secretion primarily through the activation of a basolateral, intermediate conductance Ca(2+)-sensitive K+ channel.

Exercise training improves myocardial tolerance to in vivo ischemia-reperfusion in the rat.

Experimental studies examining the effects of regular exercise on cardiac responses to ischemia and reperfusion (I/R) are limited. Therefore, these experiments examined the effects of endurance exercise training on myocardial biochemical and physiological responses during in vivo I/R. Female Sprague-Dawley rats (4 mo old) were randomly assigned to either a sedentary control group or to an exercise training group. After a 10-wk endurance exercise training program, animals were anesthetized and mechanically ventilated, and the chest was opened by thoracotomy. Coronary occlusion was achieved by a ligature around the left coronary artery; occlusion was maintained for 20 min, followed by a 10-min period of reperfusion. Compared with untrained, exercise-trained animals maintained higher (P < 0.05) peak systolic blood pressure throughout I/R. Training resulted in a significant (P < 0.05) increase in ventricular nonprotein thiols, heat shock protein (HSP) 72, and the activities of superoxide dismutase (SOD), phosphofructokinase (PFK), and lactate dehydrogenase. Furthermore, compared with untrained controls, left ventricles from trained animals exhibited lower levels (P < 0. 05) of lipid peroxidation after I/R. These data demonstrate that endurance exercise training improves myocardial contractile performance and reduces lipid peroxidation during I/R in the rat in vivo. It appears likely that the improvement in the myocardial responses to I/R was related to training-induced increases in nonprotein thiols, HSP72, and the activities of SOD and PFK in the myocardium.

Ion channels in isolated mouse jejunal crypts.

The patch-clamp technique was used to characterise the ion channels in cells located in the mid region of mouse jejunal crypts. Six different channels were seen. A large outwardly rectified K+ channel (BK) (conductance, g at 0 mV = 92 +/- 6 pS), which was highly selective for K+ [PK+ (1) > PRb+ (0.6) >> PCs+ (0.09) approximately PNa+ (0.07) > PLi+ (0.04)], had a low, voltage-independent open probability (Po) in the on-cell (O/C) configuration and appeared in 66% of the patches. In inside-out (I/O) patches, this channel had a linear current/voltage (I/V) relationship (g = 132 +/- 3 pS), Po was voltage dependent and it was blocked by cytoplasmic Ba2+ (5 mmol/l). An intermediate K+ channel (IK) which was present in 49% of O/C patches, had a linear I/V (g = 38 +/- 3 pS), ran-down in O/C patches, and was not seen in I/O patches. A number of smaller channels (SC) with conductances ranging from 5 to 20 pS were seen in 16% of O/C patches. Also present in the basolateral membrane were a Cl- channel (ICOR) and a nonselective cation channel (NSCC). These channels were only seen in I/O patches. ICOR had an outwardly rectified conductance (g at 0 mV = 36 +/- 2 pS), its Po was independent of voltage and unaffected by variations in cytoplasmic Ca2+ (100 nmol/l to 1 mmol/l) or ATP (0-1 mmol/l). The NSCC had a linear conductance (20 +/- 1 pS), its Po increased with depolarisation and elevation of cytoplasmic [Ca2+] (> or = 10 micromol/l), but was reduced by cytoplasmic ATP. None of the basolateral channels described here were activated by cAMP-dependent secretagogues, although a Cl- conductance was activated. This cAMP-dependent Cl- conductance was distinct from the basolateral Cl- channel and thus is most likely located in the apical membrane.

Yeast as a model system to study drugs effective against apicomplexan proteins.

Biochemical and genetic analyses are required to identify potential drug targets in apicomplexan parasites, but these studies have proved difficult in most parasite systems. We have developed methods based on expression of parasite proteins in the budding yeast, Saccharomyces cerevisiae, to rapidly screen drugs directed against particular parasite targets, to study the structure and function of these target molecules, and to identify mutations in the parasite genes that alter enzyme specificity or drug sensitivity. In this paper we outline the parameters that need to be considered to design yeast strains that function efficiently to assay function of parasite proteins. Basic protocols and methods are included. We detail some problems that might be encountered in the engineering of these yeast strains and suggest possible solutions.

cAMP-induced potassium channel activity in apical membrane of cultured A6 kidney cells.

In the present study, we report an adenosine 3',5'-cyclic monophosphate (cAMP)-induced potassium channel in the apical membrane of cultured A6 kidney cells grown on impermeable supports. The channel is present in approximately 10% of untreated cell-attached patches. After treatment with 1 mM dibutyryl-cAMP, the channel is present in greater than 70% of the same patches. The characteristics of this channel are 1) the channel is highly selective for potassium; 2) the channel has a unit conductance of 13 +/- 2 pS; 3) the probability of a channel opening increases in the presence of membrane permeable analogues of cAMP and with increasing depolarization of the cell interior; 4) channels are blocked by Ba2+; 5) the channel loses activity rapidly in excised patches; and 6) the channel has at least one open and two closed states. The mean open time is 3.5 +/- 1.0 ms, whereas the mean durations of the closed states are 3.2 +/- 1.4 and 29.4 +/- 3.4 ms. The channels could mediate potassium secretion in A6 cells, if the channels are normally present under transporting conditions; however, surface expression of the channels appears to depend on growth substrate and the state of cellular differentiation, since the channels are not observed in cells grown on permeable supports.

A non-selective cation channel in the apical membrane of cultured A6 kidney cells.

The patch-voltage clamp technique was used to investigate the characteristics of a non-selective cation channel (NSCC) identified in the apical membrane of cultured A6 toad kidney cells. The NSCC was present in cell-attached and inside-out membrane patches. The characteristics of this NSCC are as follows: (a) linear current-voltage relationship with a channel conductance of 21 +/- 2 pS; (b) a low selectivity between Na+ and K+ (1.5:1); (c) a high selectivity of Na+ to Cl- (greater than 45:1); (d) this channel has a single open state and two closed states; (e) the open-time constant and the second closed-time constant of this channel are voltage dependent; and (f) this NSCC is insensitive to amiloride (10(-7) M). We conclude that the NSCC resembles previously described non-selective cation channels. The NSCC of the apical membrane of A6 cells may aid in the movement of Na+ and K+ in response to varying ionic concentrations across the apical membrane.