Over-expression of plant 14-3-3 proteins in tobacco: enhancement of the plasmalemma K+ conductance of mesophyll cells.

Two cDNA clones encoding 14-3-3 homologous proteins were isolated from Vicia faba. Deduced amino acid sequences share different degrees of homology with other plant 14-3-3 proteins. Both clones, under the control of the CaMV 35S promoter, were transformed into tobacco plants. Immunoblotting showed three different forms of ca. 31, 34, and 37 kDa, indicating a covalent modification of the expressed 14-3-3 proteins. These forms were mainly present in the microsomal fraction. Patch-clamp studies of mesophyll protoplasts of the transformants revealed a strongly enhanced K+ conductance compared to the wild type. This indicates the involvement of 14-3-3 proteins in ion channel regulation, presumably by modulating kinase activities or binding the channel.

The role of proinflammatory cytokines in the generation and maintenance of joint pain.

The proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) not only promote and maintain inflammation, they also contribute to the generation and maintenance of inflammatory pain by acting at nociceptive nerve cells. A large proportion of dorsal root ganglion (DRG) neurons express TNF receptors and receptor units for stimulation with IL-6. In the rat model of antigen-induced arthritis (AIA), neutralization of TNF-alpha by etanercept and infliximab reduced inflammation-evoked mechanical hyperalgesia at the inflamed knee joint. This treatment also attenuated the infiltration of macrophages into the DRGs usually observed during the acute phase of AIA. Intra-articular application of etanercept reduced the responses of C-fibers to mechanical stimulation of the inflamed joint but did not influence responses to stimulation of the normal joint. Finally, in cultured DRG neurons TNF-alpha increased the proportion of neurons that express the TRPV1 receptor and may thus contribute to the generation of inflammation-evoked thermal hyperalgesia.

In the yeast potassium channel, Tok1p, the external ring of aspartate residues modulates both gating and conductance.

The yeast plasma-membrane potassium channel, Tok1p, is a voltage-dependent outward rectifier, the gating and steady-state conductance of which are conspicuously modulated by extracellular [K(+)] ([K(+)](o)). Activation is slow at high [K(+)](o), showing time constants (tau(a)) of approximately 90 ms when [K(+)](o) is 150 mM (depolarizing step to +100 mV), and inactivation is weak (<30%) during sustained depolarization. Lowering [K(+)](o) accelerates activation, increases peak current, and enhances inactivation, so that at 15 mM [K(+)](o) tau(a) is less than 50 ms and inactivation suppresses approximately 60% of peak current. Two negative residues, Asp292 and Asp426, near the mouth of the assembled channel, modulate both kinetics and conductance of the channel. Charge neutralization in the mutant Asp292Asn allows fast activation (tau(a) approximately 20 ms) at high [K(+)](o), peak currents diminishing with decreasing [K(+)](o), and fast, nearly complete, inactivation. The voltage dependence of tau(a) persists in the mutant, but the [K(+)](o) dependence almost disappears. Similar but smaller changes are seen in the Asp426Asn mutant, implying that pore geometry in the functional channel has twofold, not fourfold, symmetry.