Novel ion channels in the protists, Mougeotia and Saprolegnia, using sub-gigaseals.

Protoplasts of the filamentous alga, Mougeotia, and the filamentous fungal oomycete, Saprolegnia ferax, exhibit two K+ ion channels (2-6 pA) using the patch-clamp technique when the seals are less than 1 G omega (about 100 M omega). The membrane potential of the protoplasts was near 0 mV as measured intracellularly with double-barreled micropipettes; thus, inward K+ flux is due solely to concentration differences. Although conductances are in the range expected for K+ channels, the activity at 0 mV is not seen in other organisms under gigaseal conditions. This paper draws attention to the usefulness of this subsidiary patch-clamp technique and the novel characteristics of ion channels in Mougeotia and Saprolegnia.

Phytochrome activation of k channels and chloroplast rotation in mougeotia: action spectra.

The action spectra for K(+) channel activation and chloroplast rotation are shown to be similar. Both phenomena exhibit activation at 660 nanometers, inhibition at 740 nanometers, and partial activation at 460 to 500 nanometers. This confirms that K(+) channels in Mougeotia are regulated by phytochrome, and indicates that both phenomena share at least part of the same transduction pathway.

Calcium activation of mougeotia potassium channels.

Phytochrome mediates chloroplast movement in the alga Mougeotia, possibly via changes in cytosolic calcium. It is known to regulate a calcium-activated potassium channel in the algal plasma membrane. As part of a characterization of the potassium channel, we examined the properties of calcium activation. The calcium ionophore A23187 activates the channel at external [Ca(2+)] as low as 20 micromolar. However, external [Ca(2+)] is not required for activation of the channel by photoactivated phytochrome. Furthermore, when an inhibitor of calcium release from internal stores, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, hydrochloride (TMB-8), is present, red light no longer stimulates channel activity. We conclude that phytochrome activates the plasma membrane potassium channel by releasing calcium from intracellular calcium vesicles; the elevated cytosolic calcium then stimulates channel activity by an unknown mechanism. In the presence of TMB-8, red light does induce chloroplast rotation; thus, potassium channel activation may not be coupled to chloroplast rotation.

Red light regulates calcium-activated potassium channels in mougeotia plasma membrane.

The alga Mougeotia has a large central chloroplast whose positioning is regulated by photoactivation of phytochrome, possibly via modulation of cytosolic calcium (Serlin B, Roux SJ [1984] Proc Natl Acad Sci USA 81: 6368-6372). We used the patch clamp technique to examine the effects of red and far-red light on ion channel activity in the plasma membrane of Mougeotia protoplasts to determine if ion channels play a role in chloroplast movement. Patch clamping in the cell-attached mode reveals two channels of about 2 and 4 picoamperes amplitude at 0 millivolt (inside pipette) and estimated conductances of 30 and 65 picosiemens. They are activated by red light irradiation after a lag period of about 2 to 5 minutes. Far-red light, when applied immediately after red light irradiation, reverses this activation; otherwise it has no effect. This result implicates phytochrome. The addition of the calcium ionophore, A23187, also activates ion channel activity after a lag of a few minutes. The channels are not specific for calcium since they are present when calcium is removed from the external and pipette media. They are inhibited by quaternary ammonium ions. Thus, we believe they are calcium-activated potassium channels. Their possible role in chloroplast positioning is discussed.

Light-induced import of the chromoprotein, phytochrome, into mitochondria.

Mitochondria extracted from plants that were irradiated with actinic light in vivo have associated with them the chromoprotein, phytochrome. This phytochrome retains its native subunit size of 124 kDa after proteolytic treatment of the mitochondria with trypsin and chymotrypsin. This result suggests that phytochrome is not exposed on the outer surface of the outer mitochondrial membrane. Phytochrome, so protected, is not found to be associated with mitochondria derived from unirradiated plants. The possibility that the photoactivation of phytochrome induces a conformational change in its structure which facilitates its transport into the mitochondrion is discussed.

Modulation of chloroplast movement in the green alga Mougeotia by the Ca2+ ionophore A23187 and by calmodulin antagonists.

The Ca2+ ionophore A23187 can induce chloroplast rotation within a single nonirradiated Mougeotia cell. The induced turning was dependent on the position of ionophore application and Ca2+ in the external medium. The role of calmodulin in mediating light-induced chloroplast rotation in the alga Mougeotia was investigated by using the paired calmodulin-antagonist drugs W5-W7 and W12-W13. In each pair, the antagonist with the greater affinity for calmodulin had the greater inhibitor effect on the phytochrome-controlled light response. These results support the hypothesis that calcium functions as a chemical messenger to couple the stimulus of phytochrome photoactivation with physiological responses in plants.

Characterization of oat calmodulin and radioimmunoassay of its subcellular distribution.

A protein identifiable as calmodulin has been isolated from oat (Avena sativa, var Garry) tissues. This protein is relatively heat stable, binds to hydrophobic gels, and phenothiazines in a calcium-dependent fashion, and binds to antibody to rat testes calmodulin. Based on its migration on sodium dodecyl sulfate-polyacrylamide gels, ultraviolet absorption spectrum, and amino acid composition, oat calmodulin is essentially identical to calmodulin isolated from other higher plants. Radioimmunoassays indicate that calmodulin is associated with isolated oat protoplasts, mitochondria, etioplasts, and nuclei and also appears to be a component of oat cell wall fractions.

Modulation of oat mitochondrial ATPase activity by CA2+ and phytochrome.

The activity of a Mg(2+)-dependent ATPase present in highly purified preparations of Avena mitochondria was photoreversibly modulated by red/far-red light treatments. These results were obtained either with mitochondria isolated from plants irradiated with white light prior to the extraction or with mitochondria isolated from unirradiated plants only when purified phytochrome was exogenously added to the reaction mixture. Red light, which converts phytochrome to the far red-absorbing form (Pfr) depressed the ATPase activity, and far-red light reversed this effect. Addition of exogenous CaCl2 also depressed the ATPase activity, and the kinetics of inhibition were similar to the kinetics of the Pfr effects on the ATPase. The calcium chelator, ethyleneglycol-bis(beta-amino-ethyl ether)-N,N' -tetraacetic acid, blocked the effects of both CaCl2 and Pfr on the ATPase. These results are consistent with the interpretation that Pfr promotes a release of Ca2+ from the mitochondrial matrix, thereby inducing an increase in the concentration of intermembranal and extramitochondrial Ca2+.