Inhibition of an outwardly rectifying anion channel by HEPES and related buffers

Summary The effect of pH buffers and related compounds on the conductance of an outwardly rectifying anion channel has been studies using the patch-clamp technique. Single-channel current-voltage relationships were determined in solutions buffered by trace amounts of bicarbonate and in solutions containing N-substituted taurines (HEPES, MES, BES, TES) and glycines (glycylglycine, bicine and tricine), Tris andbis-Tris at millimolar concentrations. HEPES (pK_a=7.55) reduced the conductance of the channel when present on either side of the membrane. Significant inhibition was observed with 0.6mm HEPES on the cytoplasmic side (HEPES _i ) and this effect increased with [HEPES _i ] so that conductance at the reversal potential was diminished 25% with 10mm HEPES _i )and 70% at very high [HEPES _i ]. HEPES _i block was relieved by applying positive voltage but positive currents were not consistent with a Woodhulltype blocking scheme in that calculated dissociation constants and electrical distances depended on HEPES concentration. Results obtained by varying total HEPES _i concentration at constant [HEPES^–] and vice versa suggest both the anionic and zwitterionic (protonated) forms of HEPES inhibit. Structure-activity studies with related compounds indicate the sulfonate group and heterocyclic aliphatic groups are both required for inhibition from the cytoplasmic side. TES (pK_a=7.54), substituted glycine buffers (pK_a=8.1–8.4) andbis-Tris (pK_a=6.46) had no measurable effect on conductance and appear suitable for use with this channel.     

Potency of GABA at human recombinant GABAA receptors expressed in Xenopus oocytes: a mini review

GABA_A receptors are members of the ligand-gated ion channel superfamily that mediate inhibitory neurotransmission in the central nervous system. They are thought to be composed of 2 alpha (α), 2 beta (β) subunits and one other such as a gamma (γ) or delta (δ) subunit. The potency of GABA is influenced by the subunit composition. However, there are no reported systematic studies that evaluate GABA potency on a comprehensive number of subunit combinations expressed in Xenopus oocytes, despite the wide use of this heterologous expression system in structure–function studies and drug discovery. Thus, the aim of this study was to conduct a systematic characterization of the potency of GABA at 43 human recombinant GABA_A receptor combinations expressed in Xenopus oocytes using the two-electrode voltage clamp technique. The results show that the α-subunits and to a lesser extent, the β-subunits influence GABA potency. Of the binary and ternary combinations with and without the γ2L subunit, the α6/γ2L-containing receptors were the most sensitive to GABA, while the β2- or β3-subunit conferred higher sensitivity to GABA than receptors containing the β1-subunit with the exception of the α2β1γ2L and α6β1γ2L subtypes. Of the δ-subunit containing GABA_A receptors, α4/δ-containing GABA_A receptors displayed highest GABA sensitivity, with mid-nanomolar concentrations activating α4β1δ and α4β3δ receptors. At α4β2δ, GABA had low micromolar activity.     

The difference in embryo quality between Belclare and Suffolk ewes is not due to differences in oocyte quality

We have previously reported that the percentage of fertilized oocytes which reached the blastocyst stage by Day 6 after AI with frozen-thawed semen was higher for Belclare (94%) than Suffolk (59%) ewes. This may reflect differences in the timing of fertilization (Experiment 1) or differences in oocyte quality (Experiments 2 and 3). In Experiment 1, oocytes recovered from slaughterhouse ovaries were matured in vitro for 18, 20, 24, 28 or 30 h prior to fertilization and were then cultured in vitro. In Experiment 2, Belclare (n = 69) and Suffolk (n = 71) ewes were laparoscopically inseminated using frozen-thawed semen. Presumptive zygotes were recovered between 23 and 47 h post-insemination and cultured in vitro (grouped by breed). In Experiment 3, immature oocytes from Suffolk and Belclare ewes, were matured, fertilized and cultured in vitro (grouped by breed). Cleavage rate and blastocyst development was assessed. There was no effect of time of fertilization on cleavage rate, however, a lower proportion of cleaved oocytes reached the blastocyst stage after insemination at 30h compared to 24 h (P < 0.001). Ewe breed did not affect cleavage rate of oocytes matured and fertilized in vivo (41+/-9.6 and 47+/-10.1) or in vitro (47+/-9.4 and 52+/-9.4) for Belclare and Suffolk ewes, respectively (P > 0.05; %+/-S.E.). Likewise, ewe breed had no effect on the percentage (+/-S.E.) of cleaved oocytes developing to the blastocyst stage for in vivo (29+/-7.2 and 25+/-7.9) or in vitro matured and fertilized oocytes (29+/-6.1 and 36+/-5.9) from Belclare and Suffolk ewes, respectively (P>0.05). Based on this study oocyte quality does not differ between the breeds and in addition a 4h difference in the timing of fertilization, reflective of the breed difference in the timing of the LH surge in vivo, would not affect early embryo development.     

Charged Residues in Surface-located Loops Influence Voltage Gating of Porin from Haemophilus influenzae Type b

Porin of Haemophilus influenzae type b (341 amino acids; M _r 37782) determines the permeability of the outer membrane to low molecular mass compounds. Purified Hib porin was subjected to chemical modification of lysine residues by succinic anhydride. Electrospray ionization mass spectrometry identified up to 12 modifications per porin molecule. Tryptic digestion of modified Hib porin followed by reverse phase chromatography and matrix assisted laser desorption ionization time-of-flight mass spectrometry mapped the succinylation sites. Most modified lysines are positioned in surface-located loops, numbers 1 and 4 to 7. Succinylated porin was reconstituted into planar lipid bilayers, and biophysical properties were analyzed and compared to Hib porin: there was an increased average single channel conductance compared to Hib porin (1.24+/−0.41 vs. 0.85+/−0.40 nanosiemens). The voltage-gating activity of succinylated porin differed considerably from that of Hib porin. The threshold voltage for gating was decreased from 75 to 40 mV. At 80 mV, steady-state conductance for succinylated porin was 50–55% of the instantaneous conductance. Hib porin at 80 mV showed a decrease to 89–91% of the instantaneous current levels. We propose that surface-located lysine residues are determinants of voltage gating for porin of Haemophilus influenzae type b. Received: 11 August 2000/Revised: 8 September 2000     

The identification and characterization of novel PKCepsilon phosphorylation sites provide evidence for functional cross-talk within the PKC superfamily

PKCepsilon (protein kinase Cepsilon) is a phospholipid-dependent serine/threonine kinase that has been implicated in a broad array of cellular processes, including proliferation, survival, migration, invasion and transformation. Here we demonstrate that, in vitro, PKCepsilon undergoes autophosphorylation at three novel sites, Ser(234), Ser(316) and Ser(368), each of which is unique to this PKC isoform and is evolutionarily conserved. We show that these sites are phosphorylated over a range of mammalian cell lines in response to a number of different stimuli. Unexpectedly, we find that, in a cellular context, these phosphorylation events can be mediated in-trans by cPKC (classical PKC) isoforms. The functional significance of this cross-talk is illustrated through the observation that the cPKC-mediated phosphorylation of PKCepsilon at residue Ser(368) controls an established PKCepsilon scaffold interaction. Thus our current findings identify three new phosphorylation sites that contribute to the isoform-specific function of PKCepsilon and highlight a novel and direct means of cross-talk between different members of the PKC superfamily.