Summary Neuronal DEG/ENaC Na+ channels have been implicated in touch sensation. For example, MEC-4 is expressed in touch neurons in C. elegans and mediates gentle touch response. Similarly, homologous mammalian ASIC2 and ASIC3 are expressed in sensory neurons and produce touch phenotypes when knocked out in mice. Here, we show that novel DEG/ENaC subunits DELM-1 and DELM-2 are expressed in glia associated with touch neurons in C. elegans and that their knock-out causes defects in mechanosensory behaviors related to nose touch and foraging, which are mediated by OLQ and IL1 sensory neurons. Cell-specific rescue supports that DELM-1 and DELM-2 are required cell-autonomously in glia to orchestrate mechanosensory behaviors. Electron microscopy reveals that in delm-1 knockouts, OLQ and IL1 sensory neurons and associated glia are structurally normal. Furthermore, we show that knockout of DELM-1 and DELM-2 does not disrupt the expression or cellular localization of TRPA-1, a TRP channel needed in OLQ and IL1 neurons for touch behaviors. Rather, rescue of the delm-1 nose-touch insensitive phenotype by expression of a K+ channel in socket glia and of a cationic channel in OLQ neurons suggests that DELM channels set basal neuronal excitability. Taken together, our data show that DELM-1 and DELM-2 are expressed in glia associated with touch neurons where they are not needed for neuronal structural integrity or cellular distribution of neuronal sensory channels, but rather for their function.

Complementary RNA, derived from the intestine of the sea bass Dicentrarchus labrax and putatively coding for a pH-dependent oligopeptide transporter PepT1 (SLC15 family), was injected in Xenopus oocytes that were subsequently tested with electrophysiological techniques. Transport-associated currents were observed when various di- or tripeptides were applied at concentrations ranging between 0.1 and 10 mM. No currents were generated by histidine nor by other single amino acids. Sea bass PepT1 also exhibited presteady-state currents in the absence of substrates. Acidic pH slowed down the relaxation time constant of these currents and shifted both Q/V and tau/V relationships toward more positive voltages. Michaelis-Menten analysis of the transport currents showed an increase in apparent substrate affinity at acidic pH, which was very similar to that exhibited by the related transporter from zebrafish (Danio rerio), but in contrast, did not demonstrate a significant effect of pH on the maximal transport current.

Electrophysiological and biophysical analyses were used to compare the partial and complete transport cycles of the intestinal oligopeptide transporter PepT1 among three species (seabass, zebrafish and rabbit). On the whole, the presteady-state currents of the fish transporters were similar to each other. Rabbit PepT1 differed from the fish transporters by having slower-decaying currents, and the charge vs. potential (Q/V) and time constant vs. potential (τ/V) curves shifted to more positive potentials. All of the isoforms were similarly affected by external pH, showing acidity-induced slowing of the transients and positive shifts in the Q/V and τ/V curves. Analysis of the pH-dependence of the unidirectional rates of the intramembrane charge movement suggested that external protonation of the protein limits the speed of this process in both directions. The complete cycle of the transporter was studied using the neutral dipeptide Gly-Gln. Michaelis-Menten analysis confirmed that, in all species, acidity significantly increases the apparent affinity for the substrate but does not strongly impact maximal transport current. Simulations using a kinetic model incorporating the new findings showed good agreement with experimental data for all three species, both with respect to the presteady-state and the transport currents.

Synaptic glycine levels are controlled by GLYTs (glycine transporters). GLYT1 is the main regulator of synaptic glycine concentrations and catalyses Na+-Cl--glycine co-transport with a 2:1:1 stoichiometry. In contrast, neuronal GLYT2 supplies glycine to the presynaptic terminal with a 3:1:1 stoichiometry. We subjected homology models of GLYT1 and GLYT2 to molecular dynamics simulations in the presence of Na+. Using molecular interaction potential maps and in silico mutagenesis, we identified a conserved region in the GLYT2 external vestibule likely to be involved in Na+ interactions. Replacement of Asp471 in this region reduced Na+ affinity and Na+ co-operativity of transport, an effect not produced in the homologous position (Asp295) in GLYT1. Unlike the GLYT1-Asp295 mutation, this Asp471 mutant increased sodium leakage and non-stoichiometric uncoupled ion movements through GLYT2, as determined by simultaneously measuring current and [3H]glycine accumulation. The homologous Asp471 and Asp295 positions exhibited distinct cation-sensitive external accessibility, and they were involved in Na+ and Li+-induced conformational changes. Although these two cations had opposite effects on GLYT1, they had comparable effects on accessibility in GLYT2, explaining the inhibitory and stimulatory responses to lithium exhibited by the two transporters. On the basis of these findings, we propose a role for Asp471 in controlling cation access to GLYT2 Na+ sites, ion coupling during transport and the subsequent conformational changes.

Non-technical summary The oligopeptide transporter PepT1 is a protein found in the membrane of the cells of the intestinal walls, and represents the main route through which proteic nutrients are absorbed by the organism. Along the polypeptidic chain of this protein, two oppositely charged amino acids, an arginine in position 282 and an aspartate in position 341 of the sequence, have been hypothesised to form a barrier in the absorption pathway. In this paper we show that appropriate mutations of these amino acids change the properties of PepT1 in a way that confirms that these parts of the protein indeed act as an electrostatic gate in the transport process. The identification of the structural basis of the functional mechanism of this transporter is important because, in addition to its role in nutrient uptake, PepT1 represents a major pathway for the absorption of several therapeutic drugs.Abstract The effects of mutations in the charge pair residues Arg282 and Asp341 of the rabbit oligopeptide transporter PepT1 have been studied using electrophysiology in mRNA-injected Xenopus oocytes. Substitution of Arg282 with neutral or negatively charged residues produced a shift towards more positive potentials in the characteristics of charge movement with respect to the wild-type form. Conversely replacement of Asp341 with Arg reduced both pre-steady-state and transport currents and produced a negative shift of the charge movement properties. Both kinds of currents remained pH-sensitive in the mutants. All functional mutants were correctly localized on the cell membrane. Removal of the positive charge of Arg282 produced transporters able to generate conspicuous outward currents whose reversal potential was affected by external pH and by substrate concentration. This suggests that the mutants still translocate protons and substrate as a complex. Charged substrates were accepted by the mutants with the same potency order as the wild-type. The results support the idea that Arg282 and Asp341 play the role of electrostatic gates in the PepT1 transport cycle.

Hypothesis:Application of localized, mild therapeutic hypothermia during cochlear implantation (CI) surgery is feasible for residual hearing preservation.Background:CI surgery often results in a loss of residual hearing. In preclinical studies, local application of controlled, mild therapeutic hypothermia has shown promising results as a hearing preservation strategy. This study investigated a suitable surgical approach to deliver local hypothermia in patients utilizing anatomical and radiologic measurements and experimental measurements from cadaveric human temporal bones.Methods:Ten human cadaveric temporal bones were scanned with micro-computed tomography and anatomical features and measurements predicting round window (RW) visibility were characterized. For each bone, the standard facial recess and myringotomy approaches for delivery of hypothermia were developed. The St. Thomas Hospital (STH) classification was used to record degree of RW visibility with and without placement of custom hypothermia probe. Therapeutic hypothermia was delivered through both approaches and temperatures recorded at the RW, RW niche, over the lateral semicircular canal and the supero-lateral mastoid edge.Results:The average facial recess area was 13.87 ± 5.52 mm2. The introduction of the cooling probe through either approach did not impede visualization of the RW or cochleostomy as determined by STH grading. The average temperatures at RW using the FR approach reduced by 4.57 ± 1.68 °C for RW, while using the myringotomy approach reduced by 4.11 ± 0.98 °C for RW.Conclusion:Local application of therapeutic hypothermia is clinically feasible both through the facial recess and myringotomy approaches without limiting optimal surgical visualization.

Invertebrate innexin proteins share sequence homology with vertebrate pannexins and general membrane topology with both pannexins and connexins. While connexins form gap junctions that mediate intercellular communication, pannexins are thought to function exclusively as plasma membrane channels permeable to both ions and small molecules. Undoubtedly, certain innexins function as gap junction proteins. However, due to sequence similarity to pannexins, it was postulated that innexins also function as plasma membrane channels. Indeed, some of the leech innexins were found to mediate ATP release as unpaired membrane channels with shared pharmacology to pannexin channels. We show here that Caenorhabditis elegans touch-sensing neurons express a mechanically gated innexin channel with a conductance of ∼1 nS and voltage-dependent and K(+)-selective subconductance state. We also show that C. elegans touch neurons take up ethidium bromide through a mechanism that is activated and blocked by innexin activating stimuli and inhibitors, respectively. Finally, we present evidence that touch neurons' innexins are required for cell death induced by chemical ischemia. Our work demonstrates that innexins function as plasma membrane channels in native C. elegans neurons, where they may play a role in pathological cell death.

The effects of three tricyclic antidepressants (TCAs) and two serotonin selective reuptake inhibitors (SSRIs) have been studied with an electrophysiological approach on Xenopus laevis oocytes expressing the rat GABA (gamma-Aminobutyric-acid) transporter rGAT1. All tested TCAs and SSRIs inhibit the GABA-associated current in a dose-dependent way with low but comparable efficacy. The pre-steady-state and uncoupled currents appear substantially unaffected. The efficacy of desipramine, but not of the other drugs, is strongly increased in the lysine-glutamate or -aspartate mutants K448E and K448D. Comparison of I(max) and K(0.5GABA) in the absence and presence of desipramine showed that both parameters are reduced by the drug in the wild-type and in the K448E mutant. This suggests an uncompetitive inhibition, in which the drug can bind only after the substrate, an explanation in agreement with the lack of effects on the pre-steady-state and leak currents, and with the known structural data.