Functional characterisation of an intron retaining K(+) transporter of barley reveals intron-mediated alternate splicing.

Intron retention in transcripts and the presence of 5' and 3' splice sites within these introns mediate alternate splicing, which is widely observed in animals and plants. Here, functional characterisation of the K(+) transporter, HvHKT2;1, with stably retained introns from barley (Hordeum vulgare) in yeast (Saccharomyces cerevisiae), and transcript profiling in yeast and transgenic tobacco (Nicotiana tabacum) is presented. Expression of intron-retaining HvHKT2;1 cDNA (HvHKT2;1-i) in trk1, trk2 yeast strain defective in K(+) uptake restored growth in medium containing hygromycin in the presence of different concentrations of K(+) and mediated hypersensitivity to Na(+) . HvHKT2;1-i produces multiple transcripts via alternate splicing of two regular introns and three exons in different compositions. HKT isoforms with retained introns and exon skipping variants were detected in relative expression analysis of (i) HvHKT2;1-i in barley under native conditions, (ii) in transgenic tobacco plants constitutively expressing HvHKT2;1-i, and (iii) in trk1, trk2 yeast expressing HvHKT2;1-i under control of an inducible promoter. Mixed proportions of three HKT transcripts: HvHKT2;1-e (first exon region), HvHKT2;1-i1 (first intron) and HvHKT2;1-i2 (second intron) were observed. The variation in transcript accumulation in response to changing K(+) and Na(+) concentrations was observed in both heterologous and plant systems. These findings suggest a link between intron-retaining transcripts and different splice variants to ion homeostasis, and their possible role in salt stress.

Inositol hexakisphosphate is a physiological signal regulating the K+-inward rectifying conductance in guard cells.

(RS)-2-cis, 4-trans-abscisic acid (ABA), a naturally occurring plant stress hormone, elicited rapid agonist-specific changes in myo-inositol hexakisphosphate (InsP(6)) measured in intact guard cells of Solanum tuberosum (n = 5); these changes were not reproduced by (RS)-2-trans, 4-trans-abscisic acid, an inactive stereoisomer of ABA (n = 4). The electrophysiological effects of InsP(6) were assessed on both S. tuberosum (n = 14) and Vicia faba (n = 6) guard cell protoplasts. In both species, submicromolar concentrations of InsP(6), delivered through the patch electrode, mimicked the inhibitory effects of ABA and internal calcium (Ca(i)(2+)) on the inward rectifying K(+) current, I(K,in), in a dose-dependent manner. Steady state block of I(K,in) by InsP(6) was reached much more quickly in Vicia (3 min at approximately 1 microM) than Solanum (20-30 min). The effects of InsP(6) on I(K,in) were specific to the myo-inositol isomer and were not elicited by other conformers of InsP(6) (e.g., scyllo- or neo-). Chelation of Ca(2+) by inclusion of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or EGTA in the patch pipette together with InsP(6) prevented the inhibition of I(K,in), suggesting that the effect is Ca(2+) dependent. InsP(6) was approximately 100-fold more potent than Ins(1,4,5)P(3) in modulating I(K,in). Thus ABA increases InsP(6) in guard cells, and InsP(6) is a potent Ca(2+)-dependent inhibitor of I(K,in). Taken together, these results suggest that InsP(6) may play a major role in the physiological response of guard cells to ABA.

Effects of internal K+ and ABA on the voltage- and time-dependence of the outward K(+)-rectifier in Vicia guard cells.

One of the main effects of abscisic acid (ABA) is to induce net loss of potassium salts from guard cells enabling the stomata to close. K+ is released from the vacuole into the cytosol and then to the extracellular space. The effects of increasing cytosolic K+ on the voltage- and time-dependence of the outwardly rectifying K(+)-current (IK,out) in guard cell protoplasts (GCP) was examined in the whole-cell configuration of the patch-clamp technique. The same quantitative analysis was performed in the presence of ABA at different internal K+ concentrations ([K+]i). Varying [K+]i in the patch pipette from 100 to 270 mM increased the magnitude of IK,out in a nonlinear manner and caused a negative shift in the midpoint (V0.5) of its steady-state activation curve. External addition of ABA (10-20 microM) also increased the magnitude of IK,out at all [K+]i, but caused a shift in V0.5 of the steady-state activation curve only in those GCP loaded with 150 mM internal K+ or less. Indeed, V0.5 did not shift upon addition of ABA when the [K+]i was above 150 mM and up to 270 mM, i.e., the shift in V0.5 caused by ABA depended on the [K+]i. Both increase in [K+]i and external addition of ABA, decreased (by approximately 20%) the activation time constant (tau n) of IK,out. The small decrease in tau n, in both cases, was found to be independent of the membrane voltage. The results indicate that ABA mimics the effect of increasing cytoplasmic K+, and suggest that ABA may increase IK,out and alter V0.5 of its steady-state activation curve via an enhancement in cytosolic K+. This report describes for the first time the effects of [K+]i on the voltage- and time-dependence of IK,out in guard cells. It also provides an explanation for the quantitative (total membrane current) and qualitative (current kinetics) differences found between intact guard cells and their protoplasts.

Role of calcium in the modulation of Vicia guard cell potassium channels by abscisic acid: a patch-clamp study.

There is evidence for a role of increased cytoplasmic Ca2+ in the stomatal closure induced by abscisic acid (ABA), but two points of controversy remain the subject of vigorous debate--the universality of Ca2+ as a component of the signaling chain, and the source of the increased Ca2+, whether influx across the plasmalemma, or release from internal stores. We have addressed these questions by patch-clamp studies on guard cell protoplasts of Vicia faba, assessing the effects of ABA in the presence and absence of external Ca2+, and of internal Ca2+ buffers to control levels of cytoplasmic Ca2+. We show that ABA-induced reduction of the K+ inward rectifier can occur in the absence of external Ca2+, but is abolished when Ca2+ buffers are present inside the cell. Thus, some minimum level of cytoplasmic Ca2+ is a necessary component of the signaling chain by which ABA decreases the K+ inward rectifier in stomatal guard cells, thus preventing stomatal opening. Release of Ca2+ from internal stores is capable of mediating the response, in the absence of any Ca2+ influx from the extracellular medium. The work also shows that enhancement of the K+ outward rectifier by ABA is Ca2+ independent, and that other signaling mechanisms must be involved. A role for internal pH, as suggested by H.R. Irving, C.A. Gehring and R.W. Parish (Proc. Natl. Acad. Sci. USA 89:1790-1794, 1990) and M.R. Blatt (J. Gen. Physiol. 99:615-644, 1992), is an attractive working hypothesis.

Action of heptaminol hydrochloride on contractile properties in frog isolated twitch muscle fibre.

1. Heptaminol stopped or delayed the progressive decline in tension which characterizes the phenomenon of fatigue in frog isolated twitch muscle fibre. 2. Heptaminol had no action on the sodium, potassium and calcium voltage-dependent ionic conductances. 3. The hypothesis of an action via an internal alkalinization was tested by comparison with the action of NH4Cl. Both substances increased the tension. 4. The action of heptaminol was suppressed in sodium-free (TRIS) solution or in the presence of amiloride while the action of NH4Cl was always observed. 5. These results could be explained by a stimulation of the Na/H antiport by heptaminol.

Forskolin but not isoprenaline increases sodium current from frog cardiac myocytes.

The effect of forskolin on sodium current (INa) was studied in enzymatically dispersed frog atrial myocytes. The single pipette patch-clamp technique was used, with a low external sodium concentration. Forskolin (0.5 microM) increased INa at all potentials between -50 and +30 mV. The forskolin-induced increase of INa was mimicked by 1,9-dideoxyforskolin (0.5 microM) but not by isoprenaline (2 microM). These results suggest that forskolin can modulate INa through a mechanism that does not involve the production of cyclic AMP.