Application of SCAM (substituted cysteine accessibility method) to gap junction intercellular channels.

The pore-lining residues of gap junction channels determine their permeability to ions and small cellular metabolites. These residues can be identified through systematic cysteine substitution and accessibility analysis, commonly known as SCAM (Substituted Cysteine Accessibility Method). However, application of this technique to intercellular channels is more complicated than for their transmembrane counterparts. We have utilized a novel dual-oocyte perfusion device to apply cysteine reagents to the cytoplasmic face of paired, voltage-clamped Xenopus oocytes. In this configuration, a large and irreversible cysteine reagent MBB (maliemidobutyryl biocytin, mw 537) was shown to readily traverse the gap junction pore and induce conductance changes upon reaction of accessible sites. Of the 11 reactive sites identified, 6 were located in M3, where they span the bilayer. They display a periodicity characteristic of the tilted helix that lines the pore in the gap junction structure of Unger et al. (1999). Access to several of the other sites was attributed to aqueous crevices between transmembrane helices. Reactive sites were slightly different than those identified for gap junction hemichannels (Zhou et al. 1997), suggesting that conformational changes occur upon docking.

Aluminum activates an anion channel in the apical cells of wheat roots.

We describe an anion channel in the plasmalemma of protoplasts isolated from wheat (Triticum aestivum L.) roots that is activated by aluminum (Al3+). In the whole-cell configuration, addition of 20-50 microM AlCl3 to the external solution depolarized the membrane and activated an inward current that could remain active for more than 60 min. The activation by Al3+ was rapid in 20% of protoplasts examined, whereas in another 30% a delay of more than 10 min occurred after Al3+ was added. Once the current was activated, changing the external Cl- concentration shifted the membrane reversal potential with ECl, showing that the channel is more selective for anions than cations (Ca2+, K+, tetraethylammonium+). The channel could be activated by Al3+, but not by La3+, and was observed in protoplasts isolated from the root apex but not in protoplasts isolated from mature root tissue. The anion channel antagonist niflumate inhibited the current in whole cell measurements by 83% at 100 microM. Outside-out patch recordings revealed a multistate channel with single-channel conductances of between 27 and 66 pS.

Pathways for the permeation of Na+ and Cl- into protoplasts derived from the cortex of wheat roots.

Sodium permeation into cortex cells of wheat roots was examined under conditions of high external NaCI and low Ca(2+). Two types of K(+) inward rectifier were observed in some cells. The time-dependent K(+) inward rectifier was Ca(2+)-sensitive, increasing in magnitude as external Ca(2+) was decreased from 10 mM to 0.1 mM, but did not show significant permeability to Na(+). However, the spiky inward rectifier showed significant Na+ permeation at Ca(2+) concentrations of 1 and 10 mM. In cells that initially did not show K(+) inward rectifier channels, fast and sometimes slowly activating whole-cell inward currents were induced at membrane potentials negative of zero with high external Na(+) and low Ca(2+) concentrations. With 1 mM Ca(2+) in the external solution, large inward currents were carried by Rb(+), Cs(+), K(+), Li(+), and Na(+). The permeability sequence shows that K(+), Rb(+) and Cs(+) are all more permeant than Na(+), which is about equally as permeant as Li(+). When some K(+) was present with high concentrations of Na(+) the inward currents were larger than with K(+) or Na(+) alone. About 60% of the inward current was reversibly blocked when the external Ca(2+) activity was increased from 0.03 mM to 2.7 mM (half inhibition at 0.31 mM Ca(2+) activity). Changes in the characteristics of the current noise indicated that increased Ca(2+) reduced the apparent single channel amplitude. In outside-out patches inward currents were observed at membrane potentials more positive than the equilibrium potentials for K(+) and Cl(-) when the external Na(+) concentration was high. These channels were difficult to analyse but three analysis methods yielded similar conductances of about 30 pS.

Physiological effects of two FMRFamide-related peptides from the crayfish Procambarus clarkii

The present study examined the effects of two recently identified neuropeptides on crayfish hearts and on neuromuscular junctions of the crayfish deep abdominal extensor muscles. The two peptides, referred to as NF1 (Asn-Arg-Asn-Phe-Leu-Arg-Phe-NH2) and DF2 (Asp-Arg-Asn-Phe-Leu-Arg-Phe-NH2), increased the rate and amplitude of spontaneous cardiac contractions and increased the amplitude of excitatory junctional potentials (EJPs) in the deep extensors. Both effects were dose-dependent, but threshold and EC50 values for the cardiac effects were at least 10 times lower than for the deep extensor effects. The heart responded equally well to three sequential applications of peptide in any given preparation, but the responses of the deep extensors appeared to decline with successive peptide applications. The results support the hypothesis that these two neuropeptides act as neurohormones to modulate the cardiac and neuromuscular systems in crayfish. Quantal synaptic current recordings from the deep extensor muscles indicate that both peptides increase the number of quanta of transmitter released from synaptic terminals. Neither peptide elicited a measurable change in the size of quantal synaptic currents. NF1 caused a small increase in muscle cell input resistance, while DF2 did not alter input resistance. These data suggest that DF2 increases EJP amplitudes primarily by increasing transmitter release, while the increase elicited by NF1 appears to involve presynaptic and postsynaptic mechanisms.

Pump and K+ inward rectifiers in the plasmalemma of wheat root protoplasts.

An electrogenic pump, a slowly activating K+ inward rectifier and an intermittent, "spiky," K+ inward rectifier, have been identified in the plasmalemma of whole protoplasts from root cortical cells of wheat (Triticum) by the use of patch clamping techniques. Even with high external concentrations of K+ of 100 mM, the pump can maintain the membrane potential difference (PD) down to -180mV, more negative than the electrochemical equilibrium potentials of the various ions in the system. The slowly activating K+ inward rectifier, apparent in about 23% of protoplasts, allows inward current flow when the membrane PD becomes more negative than the electrochemical equilibrium potential for K+ by about 50 mV. The current usually consists of two exponentially rising components, the time constant of one about 10 times greater than the other. The longer time constant is voltage dependent, while the smaller time constant shows little voltage dependence. The rectifier deactivates, on return of the PD to less negative levels, with a single exponential time course, whose time constant is strongly voltage dependent. The spiky K+ inward rectifier, present in about 68% of protoplasts, allows intermittent current, of considerable magnitude, through the plasmalemma at PDs usually more negative than about -140mV. Patch clamp experiments on detached outside-out patches show that a possibly multi-state K+ channel, with maximum conductance greater than 400 pS, may constitute this rectifier. The paper also considers the role of the pump and the K+ inward rectifiers in physiological processes in the cell.

Temperature dependence of synaptic modulation by a FMRFamide-related neuropeptide in crayfish.

The present study examined the temperature dependence of synaptic transmission and peptidergic modulation of chemical synapses on the phasic abdominal extensor muscles of crayfish. Decreasing the temperature from 25 degrees C to 5 degrees C in saline, decreased the EPSP amplitude by 88% and increased the EPSP half-decay time four-fold. The putative neurohormone DRNFLRFamide (DF2) increased EPSP amplitudes, but was more effective at 7-9 degrees C than at 15-17 degrees C. DF2 might play a hormonal role in counteracting low transmitter release at low temperature.

Isolation of two FMRFamide-related peptides from crayfish pericardial organs.

Pericardial organs of the crayfish, Procambarus clarkii, were removed, and material from them was extracted and fractionated using two sequential reverse-phase HPLC columns. Fractions were analysed using a radioimmunoassay (RIA) specific for the C-terminal peptide sequence -Arg-Phe-NH2. The peak immunoreactive fraction from the second column appeared to contain two peptides with the sequences DRNFLRFamide and NRNFLRFamide, based on microsequencing analysis and on the absolute requirement for the amide in the RIA. These two peptides, respectively, have been given the names NF1 and DF2 due to similarity with previously identified lobster peptides F1 and F2. NF1 and DF2 are cardioexcitatory and augment synaptic transmission at a neuromuscular synapse, and evidence is presented for the calcium-dependent release of these peptides from the pericardial organs.