A Putative Calcium-Permeable Cyclic Nucleotide-Gated Channel, CNGC18, Regulates Polarized Pollen Tube Growth

A tip-focused Ca^2＋ gradient is tightly coupled to polarized pollen tube growth, and tip-localized influxes of extracellular Ca^2＋ are required for this process. However the molecular identity and regulation of the potential Ca^2＋ channels remains elusive. The present study has implicated CNGC18 （cyclic nucleotide-gated channel 18） in polarized pollen tube growth, because its overexpression induced wider and shorter pollen tubes. Moreover, CNGC18 overexpression induced depolarization of pollen tube growth was suppressed by lower extracellular calcium （[Ca^2＋]ex）. CNGC18-yellow fluorescence protein （YFP） was preferentially localized to the apparent post-Golgi vesicles and the plasma membrane （PM） in the apex of pollen tubes. The PM localization was affected by tip-localized ROP1 signaling. Expression of wild type ROP1 or an active form of ROP1 enhanced CNGC18-YFP localization to the apical region of the PM, whereas expression of RopGAP1 （a ROP1 deactivator） blocked the PM localization. These results support a role for PM-Iocalized CNGC18 in the regulation of polarized pollen tube growth through its potential function in the modulation of calcium influxes.     

Possible existence of ion pairs at the mouths of ion channels

An electrostatic calculation suggests that when an ion is bound near the mouth of a channel penetrating a low-dielectric membrane, a counter ion may form an ion pair with this ion. The tendency towards ion-pair formation is remarkably enhanced at channel mouths by forces (image forces) arising from the charges induced on the boundaries between different dielectrics. The binding constant for the formation of ion-pairs of monovalent ions is estimated under the assumption that local interactions between the counter ion and the channel wall are negligibly small. It is of the order of 1–10 molal^?1 or more for the binding of a Cl^? (F^?) counter ion to an $\text{Na}{}^{\mathrm{+}}$ ($\text{Li}{}^{\mathrm{+}}$) ion if appropriate conditions are fulfilled. The binding constant depends on the position of the binding site, the dimensions and geometries of the channel and channel mouth, and the state of ion loading of the channel, as well as the ionic species. The present results also indicate that when cation (anion) channels have anionic (cationic) groups as integrant parts of their channel walls, interactions between these charged groups and permeant ions are markedly enhanced by the image forces.     

Voltage gating in VDAC is markedly inhibited by micromolar quantities of aluminum

Summary The mitochondrial outer membrane contains voltagegated channels called VDAC that are responsible for the flux of metabolic substrates and metal ions across this membrane. The addition of micromolar quantities of aluminum chloride to phospholipid membranes containing VDAC channels greatly inhibits the voltage dependence of the channels' permeability. The channels remain in their high conducting (open) state even at high membrane potentials. An analysis of the change in the voltage-dependence parameters revealed that the steepness of the voltage dependence decreased while the voltage needed to close half the channels increased. The energy difference between the open and closed states in the absence of an applied potential did not change. Therefore, the results are consistent with aluminum neutralizing the voltage sensor of the channel. pH shift experiments showed that positively charged aluminum species in solution were not involved. The active form was identified as being either (or both) the aluminum hydroxide or the tetrahydroxoaluminate form. Both of these could reasonably be expected to neutralize a positively charged voltage sensor. Aluminum had no detectable effect of either single-channel conductance or selectivity, indicating that the sensor is probably not located in the channel proper and is distinct from the selectivity filter.     

Vanadate as an activator of ATP — sensitive potassium channels in mouse skeletal muscle

The inside-out mode of the patch-clamp technique was used to study adenosine-5-triphosphate (ATP)-sensitive K⁺ channels in mammalian skeletal muscle. Vanadate, applied to the cytoplasmic face of excised patches, was a potent activator of ATP-sensitive K⁺ channels. Divalent cations (Mg²⁺, Ca²⁺) were a prerequisite for the activating process. The maximal effect was achieved using 1 mM vanadate dissolved in Ringer, increasing the open-state probability about ninefold. The active 5 + redox form of vanadate which stimulates ATP-sensitive K⁺ channels is likely to be decavanadate V₁₀O _inf28 ^sup6– . ATP concentration-response curves have Hill coefficients near three in internal Na⁺-rich Ringer and between one and two in internal KCl solutions. Half maximal channel blockage was observed at ATP concentrations of 4 and 8 M in Ringer and KCl solutions, respectively. Internal vanadate shifted the curves towards higher ATP concentrations without affecting their slopes. Thus 50% channel blockage occurred at 65 M ATP in internal Ringer containing 0.5 mM vanadate. The results indicate that the affinity and stoichiometry of ATP binding to ATP-sensitive K⁺ channels are strongly modulated by internal cations and that the ATP sensitivity is weakened by vanadate. Offprint requests to: B. Neumcke     

Current-voltage relationships of a sodium-sensitive potassium channel in the tonoplast ofChara corallina

Summary The membrane of mechanically prepared vesicles ofChara corallina has been investigated by patch-clamp techniques. This membrane consists of tonoplast as demonstrated by the measurement of ATP-driven currents directed into the vesicles as well as by the ATP-dependent accumulation of neutral red. Addition of 1mm ATP to the bath medium induced a membrane current of about 3.2 mA·m^–2 creating a voltage across the tonoplast of about –7 mV (cytoplasmic side negative). On excised tonoplast patches, currents through single K⁺-selective channels have been investigated under various ionic conditions. The open-channel currents saturate at large voltage displacements from the equilibrium voltage for K⁺ with limiting currents of about +15 and –30 pA, respectively, as measured in symmetric 250mm KCl solutions. The channel is virtually impermeable to Na⁺ and Cl^–. However, addition of Na⁺ decreases the K⁺ currents. TheI–V relationships of the open channel as measured at various K⁺ concentrations with or without Na⁺ added are described by a 6-state model, the 12 parameters of which are determined to fit the experimental data.     

Functional renaturation of receptor polypeptides eluted from SDS polyacrylamide gels

In order to gain further support for the concept that a homo-oligomeric protein-complex may be sufficient to form a functional ligand-activated ion channel and to explore additional possibilities for the reconstitution of channel activity, a single polypeptide band of the purified neuronal AChR from insects has been electroeluted from SDS-polyacrylamide gels, the SDS removed and the polypeptides incorporated into liposomes. Liposomes were fused into planar lipid bilayers which were subsequently analysed for channel activity. Fluctuations of cation-channels were detected after addition of agonists (carbamylcholine); channel activity was blocked by antagonists (d-tubocurarine). The channels formed by electroeluted polypeptides gave conductance values, as well as kinetic data, quite similar to channels formed by the native receptor protein. Sedimentation experiments using sucrose density gradient centrifugation revealed that a considerable portion of the electroeluted polypeptides assembled during the reconstitution process to form oligomeric complexes with a sedimentation coefficient of about 10 S; thus resembling the native receptor complex. Offprint requests to: W. Hanke     

Ion channel regulation by calmodulin binding

While many ion channels are modulated by phosphorylation, there is growing evidence that they can also be regulated by Ca²⁺-calmodulin, apparently through direct binding. In some cases, this binding activates channels; in others, it modulates channel activities. These phenomena have been documented in Paramecium, in Drosophila, in vertebrate photoreceptors and olfactory receptors, as well as in ryanodine receptor Ca²⁺-release channels. Furthermore, studies on calmodulin mutants in Paramecium have shown a clear bipartite distribution of two groups of mutations in the calmodulin gene that lead to opposite behavioral and electrophysiological phenotypes. These results indicate that the N-lobe of calmodulin specifically interacts with one class of ion-channel proteins and the C-lobe with another.     

Veratridine blocks voltage-gated potassium current in human T lymphocytes and in mouse neuroblastoma cells

(i) Effects of veratridine on ionic conductances of human peripheral blood T lymphocytes have been investigated using the whole-cell patch-clamp technique, (ii) Veratridine reduces the net outward current evoked by membrane depolarizations. The reduction originates from block of a 4-aminopyridine-sensitive, voltage-gated K⁺ current, (iii) Human T lymphocytes do not appear to express voltage-gated Na⁺ channels, since inward currents are observed neither in control nor in veratridine- and bretylium-exposed lymphocytes. (iv) The effect of veratridine consists of an increase in the rate of decay of the voltage-gated K⁺ current and a reduction of the peak current amplitude. Both effects depend on veratridine concentration. Halfmaximum block occurs at 97 m and the time constant of decay is reduced by 50% at 54 m of veratridine. (v) Possible mechanisms of veratridine action are discussed. The increased rate of K⁺ current decay is most likely due to open channel block. The decrease of current amplitude may involve an additional mechanism. (vi) In cultured mouse neuroblastoma N1E-115 cells, veratridine blocks a component of voltage-gated K⁺ current, in addition to its effect on voltage-gated Na⁺ current. This result shows that the novel effect of veratridine is not confined to lymphocytes.We thank Jacobien Künzel of the Wilhelmina Hospital for Children, Utrecht, for providing the blood samples and Aart de Groot for technical assistance. The research was supported by a fellowship of the Royal Netherlands Academy of Arts and Sciences to M. Oortgiesen.