Nicotine permeates into the endoplasmic reticulum (ER) where it begins an “inside-out” pathway that leads to addiction. Shivange et al. develop genetically encoded nicotine biosensors and show that nicotine and varenicline equilibrate in the ER within seconds of extracellular application.

Kidney disease appears common in residents of Quezalguaque, Nicaragua, particularly in younger men, with features most consistent with tubulointerstitial disease. Further research is needed to elucidate the causes of kidney disease in this region.

A leucine to alanine substitution (L9ЈA) was introduced in the M2 region of the mouse ␣4 neuronal nicotinic acetylcholine receptor (nAChR) subunit. Expressed in Xenopus oocytes, ␣4(L9ЈA)␤2 nAChRs were Ն30-fold more sensitive than wild type (WT) to both ACh and nicotine. We generated knock-in mice with the L9ЈA mutation and studied their cellular responses, seizure phenotype, and sleepwake cycle. Seizure studies on ␣4-mutated animals are relevant to epilepsy research because all known mutations linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) occur in the M2 region of ␣4 or ␤2 subunits. Thalamic cultures and synaptosomes from L9ЈA mice were hypersensitive to nicotine-induced ion flux. L9ЈA mice were ϳ15-fold more sensitive to seizures elicited by nicotine injection than their WT littermates. Seizures in L9ЈA mice differed qualitatively from those in WT: L9ЈA seizures started earlier, were prevented by nicotine pretreatment, lacked EEG spike-wave discharges, and consisted of fast repetitive movements. Nicotine-induced seizures in L9ЈA mice were partial, whereas WT seizures were generalized. When L9ЈA homozygous mice received a 10 mg/kg nicotine injection, there was temporal and phenomenological separation of mutant and WT-like seizures: an initial seizure ϳ20 s after injection was clonic and showed no EEG changes. A second seizure began 3-4 min after injection, was tonic-clonic, and had EEG spike-wave activity. No spontaneous seizures were detected in L9ЈA mice during chronic video/EEG recordings, but their sleep-wake cycle was altered. Our findings show that hypersensitive ␣4* nicotinic receptors in mice mediate changes in the sleep-wake cycle and nicotineinduced seizures resembling ADNFLE.

The gene defective in cystic fibrosis encodes a C1-channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is blocked by diphenylamine-2-carboxylate (DPC) when applied extracellularly at millimolar concentrations. We studied the block of CFTR expressed in Xenopus oocytes by DPC or by a closely related molecule, flufenamic acid (FFA). Block of whole-cell CFTR currents by bath-applied DPC or by FFA, both at 200 v,M, requires several minutes to reach full effect. Blockade is voltage dependent, suggesting open-channel block: currents at positive potentials are not affected but currents at negative potentials are reduced. The binding site for both drugs senses ~ 40% of the electric field across the membrane, measured from the inside. In single-channel recordings from excised patches without blockers, the conductance was 8.0 -+ 0.4 pS in symmetric 150 mM CI-. A subconductance state, measuring ~60% of the main conductance, was often observed. Bursts to the full open state lasting up to tens of seconds were uninterrupted at depolarizing membrane voltages. At hyperpolarizing voltages, bursts were interrupted by brief closures. Either DPC or FFA (50 tzM) applied to the cytoplasmic or extracellular face of the channel led to an increase in flicker at I'm = -100 mV and not at Vm = +100 mV, in agreement with whole-cell experiments. DPC induced a higher frequency of flickers from the cytoplasmic side than the extracellular side. FFA produced longer closures than DPC; the FFA closed time was roughly equal (~1.2 ms) at -100 mV with application from either side. In cell-attached patch recordings with DPC or FFA applied to the bath, there was flickery block at Vm = -100 mV, confirming that the drugs permeate through the membrane to reach the binding site. The data are consistent with the presence of a single binding site for both drugs, reached from either end of the channel. Open-channel block by DPC or FFA may offer tools for use with site-directed mutagenesis to describe the permeation pathway.

ART treatment was not a risk factor for adverse perinatal outcome, and risks for several outcomes were somewhat lower among ART twin deliveries. Nonetheless, ART is strongly associated with twinning and twins remain a high-risk group, relative to singletons. Promoting singleton gestation in assisted conception is an important strategy for reducing adverse outcomes.

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is linked with high penetrance to several distinct nicotinic receptor (nAChR) mutations. We studied (␣4) 3 (␤2) 2 versus (␣4) 2 (␤2) 3 subunit stoichiometry for five channel-lining M2 domain mutations: S247F, S252L, 776ins3 in ␣4, V287L, and V287M in ␤2. ␣4 and ␤2 subunits were constructed with all possible combinations of mutant and wild-type (WT) M2 regions, of cyan and yellow fluorescent protein, and of fluorescent and nonfluorescent M3-M4 loops. Sixteen fluorescent subunit combinations were expressed in N2a cells. Fö rster resonance energy transfer (FRET) was analyzed by donor recovery after acceptor photobleaching and by pixel-by-pixel sensitized emission, with confirmation by fluorescence intensity ratios. Because FRET efficiency is much greater for adjacent than for nonadjacent subunits and the ␣4 and ␤2 subunits occupy specific positions in nAChR pentamers, observed FRET efficiencies from (␣4) 3 (␤2) 2 carrying fluorescent ␣4 subunits were significantly higher than for (␣4) 2 (␤2) 3 ; the converse was found for fluorescent ␤2 subunits. All tested ADNFLE mutants produced 10 to 20% increments in the percentage of intracellular (␣4) 3 (␤2) 2 receptors compared with WT subunits. In contrast, 24-to 48-h nicotine (1 M) exposure increased the proportion of (␣4) 2 (␤2) 3 in WT receptors and also returned subunit stoichiometry to WT levels for ␣4S248F and ␤2V287L nAChRs. These observations may be relevant to the decreased seizure frequency in patients with ADNFLE who use tobacco products or nicotine patches. Fluorescence-based investigations of nAChR subunit stoichiometry may provide efficient drug discovery methods for nicotine addiction or for other disorders that result from dysregulated nAChRs.Nocturnal frontal lobe epilepsy (NFLE) is marked by seizures that include rhythmic and repetitive limb movements, rapid uncoordinated movements, dystonic posturing, complex motor activities such as sleep walking and pelvic thrusting, and the elevation of the trunk and head with ictal fear and vocalization. NFLE seizures occur primarily during phase 2 of non-rapid-eye-movement sleep. They rarely progress to tonic-clonic convulsions or status epilepticus. There are often uncertain distinctions between NFLE and paroxysmal sleep disorders. The "frontal" description arises from ictal electroencephalographic data, where available, and seizure semiology origin (Provini et al., 1999;Herman et al., 2001;Combi et al., 2004;Derry et al., 2006;Ryvlin et al., 2006).Autosomal dominant NFLE (ADNFLE) (Scheffer et al., 1995) is linked, with high penetrance, to at least six distinct nAChR mutations in ␣4␤2 neuronal nicotinic acetylcholine receptors (nAChRs) (Steinlein et al., 1997;Oldani et al., 1998;Combi et al., 2004;Wimmer et al., 2009). Three mutations are in the channel-lining M2 domain of the ␣4 subunit (S247F ϭ S6ЈF in the commonly used M2 domain renumbering for Cys-loop receptors, S252L ϭ S10ЈL, and 776ins3, after the 17Ј position), whereas two mutations occur in...

We measured the permeability ratios (Px/PNa) of 3 wild-type, 1 hybrid, 2 subunit-deficient, and 22 mutant nicotinic receptors expressed in Xen0pus oocytes for alkali metal and organic cations using shifts in the biionic reversal potential of the macroscopic current. Mutations at three positions (2', 6', 10') in M2 affected ion selectivity. Mutations at position 2' (t~Thr244, 13Gly255, ~/Thr253, 8Ser258) near the intracellular end of M2 changed the organic cation permeability ratios as much as twofold and reduced Pc~/PNa and PK/PNa by 16-18%. Mutations at positions 6' and 10' increased the glycine ethyl ester/Na + and glycine methyl ester/Na + permeability ratios. Two subunit alterations also affected selectivity: omission of the 8 subunit reduced PcJPsa by 16%, and substitution ofXenopus 8 for mouse 8 increased P~nidi~ium/PNa more than twofold and reduced Pcs/PN~ by 34% and PLi/PN~ by 20%. The wild-type mouse receptor displayed a surprising interaction with the primary ammonium cations; relative permeability peaked at a chain length equal to four carbons. Analysis of the organic permeability ratios for the wild-type mouse receptor shows that (a) the diameter of the narrowest part of the pore is 8.4 ,~; (b) the mouse receptor departs significantly from size selectivity for monovalent organic cations; and (c) lowering the temperature reduces Pguanidinium/ PNa by 38% and Pbutylanunonitun/PNa more than twofold. The results reinforce present views that positions -1' and 2' are the narrowest part of the pore and suggest that positions 6' and 10' align some permeant organic cations in the pore in an interaction similar to that with channel blocker, QX-222.

Two mutations in the M2 region of the human á4 neuronal nicotinic subunit -á4(S248F) and á4(776ins3) -have been linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) (Steinlein et al. 1995(Steinlein et al. , 1997. The á4(S248F) mutation is a serine to phenylalanine substitution at position 248 in the human á4 nicotinic subunit. The á4(776ins3) mutation is a 3-base pair insertion that adds a leucine at position 259 in the amino acid sequence of the human á4 subunit. Photo-affinity labelling and structurefunction experiments show that the M2 region of the nicotinic subunits forms the conducting pore of the receptor (reviewed in Karlin & Akabas, 1995). Thus, both ADNFLE mutations lie in the conducting pore of the nicotinic receptor. ADNFLE patients suffer from brief and occasionally violent nocturnal seizures . However, the physiological mechanism responsible for these seizures has not been established. Previous studies show that the predominant brain nicotinic receptor subtype is á4â2 (Whiting & Lindstrom, 1987;Flores et al. 1991;Whiting et al. 1991). Receptors formed by co-expressing á4(S248F) or á4(776ins3) subunits with wild-type (WT) â2 subunits in Xenopus oocytes (Weiland et al. 1996; Steinlein et al. 1997;Kuryatov et al. 1997) differ from the WT receptor in several ways but no common effects of the two mutations on the acetylcholine (ACh) response have been reported previously. To determine whether the ADNFLE mutations have any common effects on the ACh response, we constructed two rat homologues (S252F and +L264) of the human ADNFLE mutations á4(S248F) and á4(777ins3), co-expressed them with rat â2 subunits in Xenopus oocytes, and studied the properties of the expressed receptors. We also constructed the rat double mutation V247I:S252F, which combined the S252F mutation with a second V247I mutation that converted the only rat/human residue substitution in the á4 M2 region to the corresponding human residue. All three Journal of Physiology (1998) 1. We constructed rat homologues (S252F and +L264) of two human á4 nicotinic mutations -á4(S248F) and á4(777ins3) -that have been linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and co-expressed them with wild-type rat â2 subunits in Xenopus oocytes. 2. The S252F and +L264 mutations had three common effects on the ACh response. First, they caused use-dependent potentiation of the response during a train of brief 100 nÒ ACh pulses. Second, they delayed the rise times of the 5-15 nÒ (+L264) and 30 nÒ (S252F) ACh responses. Third, they reduced extracellular Ca¥-induced increases in the 30 ìÒ ACh response. 3. Beside these shared effects, the S252F mutation also reduced the channel burst duration measured from voltage-jump relaxations, enhanced steady-state desensitization and reduced the single-channel conductance. In contrast, the +L264 mutation prolonged the channel burst duration, did not affect desensitization and slightly increased single-channel conductance. Neither mutation affected the number of surface receptors measured b...