Tight junctions (TJs) regulate paracellular permeability across epithelia and vary widely in their transepithelial electrical resistance (TER) and charge selectivity. The claudin family of transmembrane proteins influences these properties. We previously reported that claudin-4 increased TER approximately 300% when expressed in low-resistance Madin-Darby canine kidney (MDCK) II cells and decreased the paracellular permeability for Na(+) more than Cl(-) (Van Itallie C, Rahner C, and Anderson JM. J Clin Invest 107: 1319-1327, 2001). In comparison, we report here that expression of claudin-2 increases TER by only approximately 20% and does not change the ionic selectivity of MDCK II cells from their cation-selective background. To test whether the extracellular domains of claudins-4 and -2 determine their unique paracellular properties, we determined the effects of interchanging these domains between claudins-4 and -2. Inducible expression of wild-type claudins and extracellular domain chimeras increased both the number and depth of fibrils, but the characteristic fibril morphologies of claudin-4 or -2 were not altered by switching extracellular domains. Like claudin-4, chimeras expressing the first or both extracellular domains of claudin-4 on claudin-2 increased TER severalfold and profoundly decreased the permeability of Na(+) relative to Cl(-). In contrast, chimeras expressing the first or both extracellular domains of claudin-2 on claudin-4 increased the TER by only approximately 60 and approximately 40%, respectively, and only modestly altered charge selectivity. These results support a model in which the claudins create paracellular channels and the first extracellular domain is sufficient to determine both paracellular charge selectivity and TER.

Feeding rats diets containing oils that have a low alpha-linolenic acid [18:3(n-3)] content, such as sunflower oil, results in reduced amounts of docosahexaenoic acid [22:6(n-3)] in all brain cells and organelles compared to rats fed a diet containing soybean oil or rapeseed oil. During the period of cerebral development there is a linear relationship between the n-3 fatty acid content of the brain and that of food until alpha-linolenic acid represents approximately 200 mg/100 g food [0.4% of the total dietary energy for 18:3(n-3)]. Beyond that point brain levels reach a plateau. Similar values are also found for other organs. The level of 22:6(n-3) in membranes is little affected by the dietary quantity of linoleic acid [18:2(n-6)] if 18:3(n-3) represents approximately 0.4% of energy. In membranes from rats fed diets containing sunflower oil, Na+, K(+)-ATPase activity in nerve terminals was 60%, 5'-nucleotidase in whole brain homogenate was 80%, and 2',3'-cyclic nucleotide 3'-phosphodiesterase was 88% of that in membranes from rats fed diets containing soybean oil. A diet low in alpha-linolenic acid leads to anomalies in the electroretinogram, which partially disappear with age. It has little effect on motor activity, but it seriously affects learning tasks as measured with the shuttle box test. Rats fed a diet low in alpha-linolenic acid showed an earlier mortality in response to an intraperitoneal injection of a neurotoxin, triethyltin, than did rats fed a normal soybean oil diet.

In the heart, oxidative stress caused by exogenous H(2)O(2) has been shown to induce early afterdepolarizations (EADs) and triggered activity by impairing Na current (I(Na)) inactivation. Because H(2)O(2) activates Ca(2+)/calmodulin kinase (CaMK)II, which also impairs I(Na) inactivation and promotes EADs, we hypothesized that CaMKII activation may be an important factor in EADs caused by oxidative stress. Using the patch-clamp and intracellular Ca (Ca(i)) imaging in Fluo-4 AM-loaded rabbit ventricular myocytes, we found that exposure to H(2)O(2) (0.2 to 1 mmol/L) for 5 to 15 minutes consistently induced EADs that were suppressed by the I(Na) blocker tetrodotoxin (10 micromol/L), as well as the I(Ca,L) blocker nifedipine. H(2)O(2) enhanced both peak and late I(Ca,L), consistent with CaMKII-mediated facilitation. By prolonging the action potential plateau and increasing Ca influx via I(Ca,L), H(2)O(2)-induced EADs were also frequently followed by DADs in response to spontaneous (ie, non-I(Ca,L)-gated) sarcoplasmic reticulum Ca release after repolarization. The CaMKII inhibitor KN-93 (1 micromol/L; n=4), but not its inactive analog KN-92 (1 micromol/L, n=5), prevented H(2)O(2)-induced EADs and DADs, and the selective CaMKII peptide inhibitor AIP (autocamtide-2-related inhibitory peptide) (2 micromol/L) significantly delayed their onset. In conclusion, H(2)O(2)-induced afterdepolarizations depend on both impaired I(Na) inactivation to reduce repolarization reserve and enhancement of I(Ca,L) to reverse repolarization, which are both facilitated by CaMKII activation. Our observations support a link between increased oxidative stress, CaMKII activation, and afterdepolarizations as triggers of lethal ventricular arrhythmias in diseased hearts.

A network of oscillatory bursting neurons with excitatory coupling is hypothesized to define the primary kernel for respiratory rhythm generation in the pre-Bötzinger complex (pre-BötC) in mammals. Two minimal models of these neurons are proposed. In model 1, bursting arises via fast activation and slow inactivation of a persistent Na+ current INaP-h. In model 2, bursting arises via a fast-activating persistent Na+ current INaP and slow activation of a K+ current IKS. In both models, action potentials are generated via fast Na+ and K+ currents. The two models have few differences in parameters to facilitate a rigorous comparison of the two different burst-generating mechanisms. Both models are consistent with many of the dynamic features of electrophysiological recordings from pre-BötC oscillatory bursting neurons in vitro, including voltage-dependent activity modes (silence, bursting, and beating), a voltage-dependent burst frequency that can vary from 0.05 to >1 Hz, and a decaying spike frequency during bursting. These results are robust and persist across a wide range of parameter values for both models. However, the dynamics of model 1 are more consistent with experimental data in that the burst duration decreases as the baseline membrane potential is depolarized and the model has a relatively flat membrane potential trajectory during the interburst interval. We propose several experimental tests to demonstrate the validity of either model and to differentiate between the two mechanisms.

Using a combination of data base searching, polymerase chain reaction, and library screening, we have identified a putative K-Cl cotransporter isoform (KCC2) in rat brain that is specifically localized in neurons. A cDNA of 5566 bases was obtained from overlapping clones and encoded a protein of 1116 amino acids with a deduced molecular mass of 123.6 kDa. Over its full length, the amino acid sequence of KCC2 is 67% identical to the widely distributed K-Cl cotransporter isoform (KCC1) identified in rat brain and rabbit kidney (Gillen, C., Brill, S., Payne, J.A., and Forbush, B., III(1996) J. Biol. Chem. 271, 16237-16244) but only approximately25% identical to other members of the cation-chloride cotransporter gene family, including "loop" diuretic-sensitive Na-K-Cl cotransport and thiazide-sensitive Na-Cl cotransport. Based on analysis of the primary structure as well as homology with other cation-chloride cotransporters, we predict 12 transmembrane segments bounded by N- and C-terminal cytoplasmic regions. Four sites for N-linked glycosylation are predicted on an extracellular intermembrane loop between putative transmembrane segments 5 and 6. Northern blot analysis using a KCC2-specific cDNA probe revealed a very highly expressed approximately5.6-kilobase transcript only in brain. Reverse transcriptase-polymerase chain reaction revealed that KCC1 was present in rat primary astrocytes and rat C6 glioma cells but that KCC2 was completely absent from these cells, suggesting KCC2 was not of glial cell origin. In situ hybridization studies demonstrated that the KCC2 transcript was expressed at high levels in neurons throughout the central nervous system, including CA1-CA4 pyramidal neurons of the hippocampus, granular cells and Purkinje neurons of the cerebellum, and many groups of neurons throughout the brainstem.

In early stages of permanent renal injury or extensive ablation, structural and functional adaptations associated with hypertrophy partially compensate for nephron losses. Glomerulotubular balance is maintained in these conditioned nephrons by intrinsic tubule and peritubular capillary adaptations that parallel single nephron glomerular filtration rate (SNGFR). Studies of Na+-H+ exchange in renal cortical brush border membrane vesicles indicate that tubule functional adaptation is not tied to loss of renal mass per se but rather to factors such as dietary protein content that set the level of SNGFR. Likewise, the structural heterogeneity that follows chronic renal injury or extreme ablation of renal mass is less a consequence of nephron injury than of adaptation linked to dietary protein intake. Indeed, since dietary protein restriction blunts the need for compensatory glomerular hyperfiltration, there is neither a stimulus for nephron hypertrophy nor for enhanced tubule ion and fluid transport. In rats with remnant kidneys, experimentally induced diabetes mellitus, or severe hypertension, increases in glomerular pressures and flows precede proteinuria, glomerular sclerosis, and azotemia. Protein restriction prevents these hemodynamic adaptations as well as the late complications. Similar conclusions appear to be applicable to a wide spectrum of clinical circumstances characterized by reduced nephron number.

Multiple sclerosis is the most common cause of non-traumatic neurological impairment in young adults. An energy deficient state has been implicated in the degeneration of axons, the pathological correlate of disease progression, in multiple sclerosis. Mitochondria are the most efficient producers of energy and play an important role in calcium homeostasis. We analysed the density and function of mitochondria using immunohistochemistry and histochemistry, respectively, in chronic active and inactive lesions in progressive multiple sclerosis. As shown before in acute pattern III and Balo's lesions, the mitochondrial respiratory chain complex IV activity is reduced despite the presence of mitochondria in demyelinated axons with amyloid precursor protein accumulation, which are predominantly located at the active edge of chronic active lesions. Furthermore, the strong non-phosphorylated neurofilament (SMI32) reactivity was associated with a significant reduction in complex IV activity and mitochondria within demyelinated axons. The complex IV defect associated with axonal injury may be mediated by soluble products of innate immunity, as suggested by an inverse correlation between complex IV activity and macrophage/microglial density in chronic lesions. However, in inactive areas of chronic multiple sclerosis lesions the mitochondrial respiratory chain complex IV activity and mitochondrial mass, judged by porin immunoreactivity, are increased within approximately half of large (>2.5 microm diameter) chronically demyelinated axons compared with large myelinated axons in the brain and spinal cord. The axon-specific mitochondrial docking protein (syntaphilin) and phosphorylated neurofilament-H were increased in chronic lesions. The lack of complex IV activity in a proportion of Na(+)/K(+) ATPase alpha-1 positive demyelinated axons supports axonal dysfunction as a contributor to neurological impairment and disease progression. Furthermore, in vitro studies show that inhibition of complex IV augments glutamate-mediated axonal injury (amyloid precursor protein and SMI32 reactivity). Our findings have important implications for both axonal degeneration and dysfunction during the progressive stage of multiple sclerosis.

We sought to determine whether low-affinity, high-capacity mitochondrial Ca2+ uptake contributes to buffering physiological Ca2+ loads in sensory neurons. Intracellular free calcium concentration ([Ca2+]i) and intracellular free hydrogen ion concentration ([H+]i) were measured in single rat dorsal root ganglion (DRG) neurons grown in primary culture using indo-1 and carboxy-SNARF-based dual emission microfluorimetry. Field potential stimulation evoked action potential-mediated increases in [Ca2+]. Brief trains of action potentials elicited [Ca2+]i transients that recovered to basal levels by a single exponential process. Trains of>> 25 action potentials elicited larger increases in [Ca2+]i, recovery from which consisted of three distinct phases. During a rapid initial phase [Ca2+]i decreased to a plateau level (450-550 nM). The plateau was followed by a slow return to basal [Ca2+]i [Ca2+]i transients elicited by 40-50 action potentials in the presence of the mitochondrial uncoupler carbonyl cyanide chlorophenyl hydrazone (CCCP), or the electron transport inhibitor antimycin A1, lacked the plateau, and the recovery to basal [Ca2+]i consisted of a single slow phase. Depolarization with 50 mM K+ produced a multiphasic [Ca2+]i transient and increased [H+]i from 74 +/- 3 to 107 +/- 8 nM. The rise in [H+]i was dependent upon extracellular Ca2+ and was inhibited by mitochondrial poisons. With mitochondrial Ca2+ buffering pharmacologically blocked, the recovery to basal [Ca2+]i was unaffected by removal of extracellular Na+. We conclude that large Ca2+ loads are initially buffered by fast mitochondrial sequestration that effectively uncouples electron transport from ATP synthesis, leading to an increase in [H+]i. Small Ca2+ loads are buffered by a nonmitochondrial, Na(+)-independent process.

We have examined the distribution of the sensory neuron-specific Na+ channel Nav1.8 (SNS/PN3) in nociceptive and non-nociceptive dorsal root ganglion (DRG) neurons and whether its distribution is related to neuronal membrane properties. Nav1.8-like immunoreactivity (Nav1.8-LI) was examined with an affinity purified polyclonal antiserum (SNS11) in rat DRG neurons that were classified according to sensory receptive properties and by conduction velocity (CV) as C-, Adelta- or Aalpha/beta. A significantly higher proportion of nociceptive than low threshold mechanoreceptive (LTM) neurons showed Nav1.8-LI, and nociceptive neurons had significantly more intense immunoreactivity in their somata than LTM neurons. Results showed that 89, 93 and 60% of C-, Adelta- and Aalpha/beta-fibre nociceptive units respectively and 88% of C-unresponsive units were positive. C-unresponsive units had electrical membrane properties similar to C-nociceptors and were considered to be nociceptive-type neurons. Weak positive Nav1.8-LI was also present in some LTM units including a C LTM, all Adelta LTM units (D hair), about 10% of cutaneous LTM Aalpha/beta-units, but no muscle spindle afferent units. Nav1.8-LI intensity was negatively correlated with soma size (all neurons) and with dorsal root CVs in A- but not C-fibre neurons. Nav1.8-LI intensity was positively correlated with action potential (AP) duration (both rise and fall time) in A-fibre neurons and with AP rise time only in positive C-fibre neurons. It was also positively correlated with AP overshoot in positive neurons. Thus high levels of Nav1.8 protein may contribute to the longer AP durations (especially in A-fibre neurons) and larger AP overshoots that are typical of nociceptors.

Point mutations can generate defective and sometimes harmful proteins. The nonsense-mediated mRNA decay (NMD) pathway minimizes the potential damage caused by nonsense mutations. In-frame nonsense codons located at a minimum distance upstream of the last exon-exon junction are recognized as premature termination codons (PTCs), targeting the mRNA for degradation. Some nonsense mutations cause skipping of one or more exons, presumably during pre-mRNA splicing in the nucleus; this phenomenon is termed nonsense-mediated altered splicing (NAS), and its underlying mechanism is unclear. By analyzing NAS in BRCA1, we show here that inappropriate exon skipping can be reproduced in vitro, and results from disruption of a splicing enhancer in the coding sequence. Enhancers can be disrupted by single nonsense, missense and translationally silent point mutations, without recognition of an open reading frame as such. These results argue against a nuclear reading-frame scanning mechanism for NAS. Coding-region single-nucleotide polymorphisms (cSNPs) within exonic splicing enhancers or silencers may affect the patterns or efficiency of mRNA splicing, which may in turn cause phenotypic variability and variable penetrance of mutations elsewhere in a gene.

K channels of bovine adrenal chromaffin cells were studied using patch-clamp techniques. Whole-cell K currents measured near +10 mV were much larger in 1 mM-external Ca than in Ca-free saline. Noise analysis suggested that this Ca-dependent current was carried by a large unitary conductance channel, called BK channel, which was previously described in inside-out patches (Marty, 1981). The Ca-dependent K current near +10 mV declined with time due to 'run-down' of Ca channels. At the same time, a fraction of the outward current observed above +50 mV was also eliminated. This outward current component probably represents K efflux through Ca channels. Whole-cell Ca-dependent K currents were studied using various Ca buffers. EGTA buffers were surprisingly inefficient: in order to block the current entirely, it was necessary to use an isotonic EGTA solution and to increase internal pH. 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was at least five times more efficient than EGTA. In isolated patches three types of single-channel K currents were observed. Under normal ionic conditions (140 mM-K inside, 140 mM-Na outside), the unitary conductances measured between -20 and +40 mV were 96 pS, 18 pS and 8 pS. The 96 pS channels are the Ca-dependent BK channels. 18 pS and 8 pS channels were both activated and then inactivated by membrane depolarization. Both displayed complex kinetics; single-channel currents were grouped in bursts. Activation and inactivation kinetics were faster for the 18 pS channel (therefore termed FK channel, for fast K channel) than for the 8 pS channel (SK channel, for slow or small amplitude channel). The voltage dependence of opening probability was steeper for the FK channel as compared to the SK channel.

The contractile response of the guinea-pig vas deferens to tetanic nerve stimulation was biphasic. The first phase was mimicked by exogenously applied ATP. The second more tonic phase was mimicked by exogenously applied noradrenaline (NA). Intracellular micro-electrodes were used to record the electrical response of the vas deferens to nerve stimulation and to exogenously applied ATP and NA. Local application of ATP (10(-5) to 10(-3)M), by pressure ejection from a micropipette, produced a depolarization similar in magnitude and time course to the excitatory junction potential (e.j.p.). NA produced no such response. Superfusion of the vas deferens with ATP and NA (10(-6) to 10(-4)M) produced a depolarization. The depolarization produced by NA was more gradual than that produced by the same concentration of ATP. The ATP-receptor antagonist ANAPP3 (arylazido aminopropionyl-ATP) preferentially antagonized the first component of the neurogenic contractile response and also antagonized the e.j.p. The alpha-receptor antagonist prazosin preferentially antagonized the second phase of the neurogenic contractile response and enhanced the e.j.p. Similar results were obtained using the irreversible alpha-receptor antagonists phenoxybenzamine and dibenamine. Cocaine (10(-6) and 10(-5)M) enhanced the second phase of the contractile response to nerve stimulation, but reduced the first phase. Lidocaine (10(-5) and 10(-4)M) had no such effect. Cocaine (10(-6) and 10(-5)M) reduced the magnitude of e.j.p.s. at all stimulation frequencies from 1 to 8 Hz. In the presence of the selective alpha 2-receptor antagonist yohimbine (10(-7)M), both phases of the contractile response to nerve stimulation were enhanced to the same degree. This concentration of yohimbine also increased the magnitude of e.j.p.s. In the presence of 10(-7) M-yohimbine, cocaine (10(-6) and 10(-5)M) still enhanced the second phase of the contractile response, but no longer reduced the initial phase of the contraction or e.j.p.s to the same degree. In vas deferens from animals pre-treated with reserpine (2 mg/kg.day), the second phase of the contractile response to nerve stimulation was reduced but neither the first phase of the contraction nor the e.j.p.s was blocked. These results suggest that the first phase of the neurogenic contractile response of the vas deferens and the e.j.p. are mediated by ATP acting on P2-purinoreceptors, whereas NA mediates phase two, via alpha 1-adrenoceptors. The results also suggest that release of ATP and NA is influenced by a negative feed-back mechanism involving presynaptic alpha 2-adrenoceptors.

For influenza virus, we developed an efficient, noncytotoxic, plasmid-based virus-like particle (VLP) system to reflect authentic virus particles. This system was characterized biochemically by analysis of VLP protein composition, morphologically by electron microscopy, and functionally with a VLP infectivity assay. The VLP system was used to address the identity of the minimal set of viral proteins required for budding. Combinations of viral proteins were expressed in cells, and the polypeptide composition of the particles released into the culture media was analyzed. Contrary to previous findings in which matrix (M1) protein was considered to be the driving force of budding because M1 was found to be released copiously into the culture medium when M1 was expressed by using the vaccinia virus T7 RNA polymerase-driven overexpression system, in our noncytotoxic VLP system M1 was not released efficiently into the culture medium. Additionally, hemagglutinin (HA), when treated with exogenous neuraminidase (NA) or coexpressed with viral NA, could be released from cells independently of M1. Incorporation of M1 into VLPs required HA expression, although when M1 was omitted from VLPs, particles with morphologies similar to those of wild-type VLPs or viruses were observed. Furthermore, when HA and NA cytoplasmic tail mutants were included in the VLPs, M1 failed to be efficiently incorporated into VLPs, consistent with a model in which the glycoproteins control virus budding by sorting to lipid raft microdomains and recruiting the internal viral core components. VLP formation also occurred independently of the function of Vps4 in the multivesicular body pathway, as dominant-negative Vps4 proteins failed to inhibit influenza VLP budding.

The epithelial Na+ channel (ENaC) forms the pathway for Na+ absorption across epithelia, including the kidney collecting duct, where it plays a critical role in Na+ homeostasis and blood pressure control. Na+ absorption is regulated in part by mechanisms that control the expression of ENaC at the apical cell surface. Nedd4 family members (e.g. Nedd4, Nedd4-2) bind to the channel and decrease its surface expression by catalyzing its ubiquitination and degradation. Conversely, serum and glucocorticoid-regulated kinase (SGK), a downstream mediator of aldosterone, increases the expression of ENaC at the cell surface. Here we show that SGK and human Nedd4-2 (hNedd4-2) converge in a common pathway to regulate epithelial Na+ absorption. Consistent with this model, we found that SGK bound to hNedd4-2 and hNedd4. A PY motif in SGK mediated the interaction and was required for SGK to stimulate ENaC. SGK phosphorylated hNedd4-2 (but not hNedd4), altering hNedd4-2 function; phosphorylation reduced the binding of hNedd4-2 to alphaENaC, and hence, the hNedd4-2-mediated inhibition of Na+ absorption. These data suggest that SGK regulates epithelial Na+ absorption in part by modulating the function of hNedd4-2.

Deprivation of afferent inputs in neural circuits leads to diverse plastic changes in both pre- and postsynaptic elements that restore neural activity. The axon initial segment (AIS) is the site at which neural signals arise, and should be the most efficient site to regulate neural activity. However, none of the plasticity currently known involves the AIS. We report here that deprivation of auditory input in an avian brainstem auditory neuron leads to an increase in AIS length, thus augmenting the excitability of the neuron. The length of the AIS, defined by the distribution of voltage-gated Na(+) channels and the AIS anchoring protein, increased by 1.7 times in seven days after auditory input deprivation. This was accompanied by an increase in the whole-cell Na(+) current, membrane excitability and spontaneous firing. Our work demonstrates homeostatic regulation of the AIS, which may contribute to the maintenance of the auditory pathway after hearing loss. Furthermore, plasticity at the spike initiation site suggests a powerful pathway for refining neuronal computation in the face of strong sensory deprivation.

Noble-metal nanocages comprise a novel class of nanostructures possessing hollow interiors and porous walls. They are prepared using a remarkably simple galvanic replacement reaction between solutions containing metal precursor salts and Ag nanostructures prepared through polyol reduction. The electrochemical potential difference between the two species drives the reaction, with the reduced metal depositing on the surface of the Ag nanostructure. In our most studied example, involving HAuCl(4) as the metal precursor, the resultant Au is deposited epitaxially on the surface of the Ag nanocubes, adopting their underlying cubic form. Concurrent with this deposition, the interior Ag is oxidized and removed, together with alloying and dealloying, to produce hollow and, eventually, porous structures that we commonly refer to as Au nanocages. This approach is versatile, with a wide range of morphologies (e.g., nanorings, prism-shaped nanoboxes, nanotubes, and multiple-walled nanoshells or nanotubes) available upon changing the shape of the initial Ag template. In addition to Au-based structures, switching the metal salt precursors to Na(2)PtCl(4) and Na(2)PdCl(4) allows for the preparation of Pt- and Pd-containing hollow nanostructures, respectively. We have found that changing the amount of metal precursor added to the suspension of Ag nanocubes is a simple means of tuning both the composition and the localized surface plasmon resonance (LSPR) of the metal nanocages. Using this approach, we are developing structures for biomedical and catalytic applications. Because discrete dipole approximations predicted that the Au nanocages would have large absorption cross-sections and because their LSPR can be tuned into the near-infrared (where the attenuation of light by blood and soft tissue is greatly reduced), they are attractive materials for biomedical applications in which the selective absorption of light at great depths is desirable. For example, we have explored their use as contrast enhancement agents for both optical coherence tomography and photoacoustic tomography, with improved performance observed in each case. Because the Au nanocages have large absorption cross-sections, they are also effective photothermal transducers; thus, they might provide a therapeutic effect through selective hyperthermia-induced killing of targeted cancer cells. Our studies in vitro have illustrated the feasibility of applying this technique as a less-invasive form of cancer treatment.

Primary aldosteronism is the most prevalent form of secondary hypertension. To explore molecular mechanisms of autonomous aldosterone secretion, we performed exome sequencing of aldosterone-producing adenomas (APAs). We identified somatic hotspot mutations in the ATP1A1 (encoding an Na(+)/K(+) ATPase α subunit) and ATP2B3 (encoding a Ca(2+) ATPase) genes in three and two of the nine APAs, respectively. These ATPases are expressed in adrenal cells and control sodium, potassium and calcium ion homeostasis. Functional in vitro studies of ATP1A1 mutants showed loss of pump activity and strongly reduced affinity for potassium. Electrophysiological ex vivo studies on primary adrenal adenoma cells provided further evidence for inappropriate depolarization of cells with ATPase alterations. In a collection of 308 APAs, we found 16 (5.2%) somatic mutations in ATP1A1 and 5 (1.6%) in ATP2B3. Mutation-positive cases showed male dominance, increased plasma aldosterone concentrations and lower potassium concentrations compared with mutation-negative cases. In summary, dominant somatic alterations in two members of the ATPase gene family result in autonomous aldosterone secretion.

At least 5% of individuals with hypertension have adrenal aldosterone-producing adenomas (APAs). Gain-of-function mutations in KCNJ5 and apparent loss-of-function mutations in ATP1A1 and ATP2A3 were reported to occur in APAs. We find that KCNJ5 mutations are common in APAs resembling cortisol-secreting cells of the adrenal zona fasciculata but are absent in a subset of APAs resembling the aldosterone-secreting cells of the adrenal zona glomerulosa. We performed exome sequencing of ten zona glomerulosa-like APAs and identified nine with somatic mutations in either ATP1A1, encoding the Na(+)/K(+) ATPase α1 subunit, or CACNA1D, encoding Cav1.3. The ATP1A1 mutations all caused inward leak currents under physiological conditions, and the CACNA1D mutations induced a shift of voltage-dependent gating to more negative voltages, suppressed inactivation or increased currents. Many APAs with these mutations were <1 cm in diameter and had been overlooked on conventional adrenal imaging. Recognition of the distinct genotype and phenotype for this subset of APAs could facilitate diagnosis.

Short-chain fatty acids (SCFAs) constitute the major solute fraction of normal stool water and are responsible for the diarrhea associated with carbohydrate (CHO) malabsorption. Although SCFA absorption from the human small bowel has been reported previously, the fate of SCFAs in the colon--their major site of production--was investigated in the present study. The colon of normal volunteers was perfused with neutral, isotonic solutions containing SCFA, 0-90 mM. Propionate was studied in detail with limited observations on acetate and n-butyrate. SCFA absorption was concentration-dependent; back diffusion of metabolic products, ketone bodies, was quantitatively insignificant. The transport process was accompanied by increased Na, K, and water absorption, by luminal alkalinization due to bicarbonate accumulation, and by a fall in lumen PCO2. The results are consistent with the existence of two mechanisms for colonic SCFA absorption: first, nonionic diffusion of protonated SCFA involving consumption of luminal CO2; this process accounts for about 60% of total SCFA absorption; and second, cellular uptake by ionic diffusion of the Na or K salt of the SCFA.

OBJECTIVE

Designer β-keto amphetamines (e.g. cathinones, 'bath salts' and 'research chemicals') have become popular recreational drugs, but their pharmacology is poorly characterized.

METHODS

We determined the potencies of cathinones to inhibit DA, NA and 5-HT transport into transporter-transfected HEK 293 cells, DA and 5-HT efflux from monoamine-preloaded cells, and monoamine receptor binding affinity.

RESULTS

Mephedrone, methylone, ethylone, butylone and naphyrone acted as non-selective monoamine uptake inhibitors, similar to cocaine. Mephedrone, methylone, ethylone and butylone also induced the release of 5-HT, similar to 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) and other entactogens. Cathinone, methcathinone and flephedrone, similar to amphetamine and methamphetamine, acted as preferential DA and NA uptake inhibitors and induced the release of DA. Pyrovalerone and 3,4-methylenedioxypyrovalerone (MDPV) were highly potent and selective DA and NA transporter inhibitors but unlike amphetamines did not evoke the release of monoamines. The non-β-keto amphetamines are trace amine-associated receptor 1 ligands, whereas the cathinones are not. All the cathinones showed high blood-brain barrier permeability in an in vitro model; mephedrone and MDPV exhibited particularly high permeability.

CONCLUSIONS

Cathinones have considerable pharmacological differences that form the basis of their suggested classification into three groups. The predominant action of all cathinones on the DA transporter is probably associated with a considerable risk of addiction.

The light-gated cation channel channelrhodopsin-2 (ChR2) has rapidly become an important tool in neuroscience, and its use is being considered in therapeutic interventions. Although wild-type and known variant ChR2s are able to drive light-activated spike trains, their use in potential clinical applications is limited by either low light sensitivity or slow channel kinetics. We present a new variant, calcium translocating channelrhodopsin (CatCh), which mediates an accelerated response time and a voltage response that is ~70-fold more light sensitive than that of wild-type ChR2. CatCh's superior properties stem from its enhanced Ca²(+) permeability. An increase in [Ca²(+)](i) elevates the internal surface potential, facilitating activation of voltage-gated Na(+) channels and indirectly increasing light sensitivity. Repolarization following light-stimulation is markedly accelerated by Ca²(+)-dependent BK channel activation. Our results demonstrate a previously unknown principle: shifting permeability from monovalent to divalent cations to increase sensitivity without compromising fast kinetics of neuronal activation. This paves the way for clinical use of light-gated channels.

To search for novel transcriptional pathways that are activated in skeletal muscle after endurance exercise, we used cDNA microarrays to measure global mRNA expression after an exhaustive bout of high-intensity cycling (approximately 75 min). Healthy, young, sedentary males performed the cycling bout, and skeletal muscle biopsies were taken from the vastus lateralis before, and at 3 and 48 h after exercise. We examined mRNA expression in individual muscle samples from four subjects using cDNA microarrays, used repeated-measures significance analysis of microarray (SAM) to determine statistically significant expression changes, and confirmed selected results using real-time RT-PCR. In total, the expression of 118 genes significantly increased 3 h postcycling and 8 decreased. At 48 h, the expression of 29 genes significantly increased and 5 decreased. Many of these are potentially important novel genes involved in exercise recovery and adaptation, including several involved in 1) metabolism and mitochondrial biogenesis (FOXO1, PPARdelta, PPARgamma, nuclear receptor binding protein 2, IL-6 receptor, ribosomal protein L2, aminolevulinate delta-synthase 2); 2) the oxidant stress response (metalothioneins 1B, 1F, 1G, 1H, 1L, 2A, 3, interferon regulatory factor 1); and 3) electrolyte transport across membranes [Na+-K+-ATPase (beta3), SERCA3, chloride channel 4]. Others include genes involved in cell stress, proteolysis, apoptosis, growth, differentiation, and transcriptional activation, as well as all three nuclear receptor subfamily 4A family members (Nur77, Nurr1, and Nor1). This study is the first to characterize global mRNA expression during recovery from endurance exercise, and the results provide potential insight into 1) the transcriptional contributions to homeostatic recovery in human skeletal muscle after endurance exercise, and 2) the transcriptional contributions from a single bout of endurance exercise to the adaptive processes that occur after a period of endurance exercise training.

1. A long-lasting afterhyperpolarization (AHP) follows current-induced repetitive firing in hippocampal CA1 neurons studied in vitro. A 10-25% increase in membrane slope conductance occurs during the AHP, suggesting that it may be mediated by an increased conductance to either K+ or Cl-. 2. Intracellular Cl- iontophoresis does not alter the AHP but does attenuate the IPSP. In contrast Ba2+, a cation that can decrease K+ conductance, eliminates the AHP but not the IPSP. These findings suggest the AHP is produced by a long-lasting increased conductance to K+, and is distinct from the IPSP. 3. Mn2+, a Ca2+-channel blocker, eliminates the AHP. In comparison, the AHP persists in the presence of the Na+-channel blocker, tetrodotoxin (TTX), and appears to be temporally associated with TTX-resistant "Ca2+ spikes." It is concluded that AHP is probably activated by Ca2+ influx. 4. These observations indicate that the AHP may be produced by a Ca2+ activated K+ current. A balance between cellular depolarization produced by Ca2+ entry and repolarization generated by a Ca2+-activated K+ current appears to operate to control excitability in some mammalian cortical neurons as it does in molluscan neurons. Disruption of this balance by Ba2+ produces spontaneous membrane-potential oscillations and recurrent burst firing in hippocampal neurons. Increases in the magnitude and duration of Ca2+ depolarization and/or decreases in the Ca2+-activated, K+-mediated repolarization may be mechanisms that lead to spontaneous, epileptiform bursting in mammalian cortical neurons.

Mutations in the serine-threonine kinases WNK1 and WNK4 [with no lysine (K) at a key catalytic residue] cause pseudohypoaldosteronism type II (PHAII), a Mendelian disease featuring hypertension, hyperkalemia, hyperchloremia, and metabolic acidosis. Both kinases are expressed in the distal nephron, although the regulators and targets of WNK signaling cascades are unknown. The Cl(-) dependence of PHAII phenotypes, their sensitivity to thiazide diuretics, and the observation that they constitute a "mirror image" of the phenotypes resulting from loss of function mutations in the thiazide-sensitive Na-Cl cotransporter (NCCT) suggest that PHAII may result from increased NCCT activity due to altered WNK signaling. To address this possibility, we measured NCCT-mediated Na(+) influx and membrane expression in the presence of wild-type and mutant WNK4 by heterologous expression in Xenopus oocytes. Wild-type WNK4 inhibits NCCT-mediated Na-influx by reducing membrane expression of the cotransporter ((22)Na-influx reduced 50%, P < 1 x 10(-9), surface expression reduced 75%, P < 1 x 10(-14) in the presence of WNK4). This inhibition depends on WNK4 kinase activity, because missense mutations that abrogate kinase function prevent this effect. PHAII-causing missense mutations, which are remote from the kinase domain, also prevent inhibition of NCCT activity, providing insight into the pathophysiology of the disorder. The specificity of this effect is indicated by the finding that WNK4 and the carboxyl terminus of NCCT coimmunoprecipitate when expressed in HEK 293T cells. Together, these findings demonstrate that WNK4 negatively regulates surface expression of NCCT and implicate loss of this regulation in the molecular pathogenesis of an inherited form of hypertension.