A vacuolar phosphate transporter essential for phosphate homeostasis in Arabidopsis

Inorganic phosphate (Pi) is stored in the vacuole, allowing plants to adapt to variable Pi availability in the soil. The transporters that mediate Pi sequestration into vacuole remain unknown, however. Here we report the functional characterization of Vacuolar Phosphate Transporter 1 (VPT1), an SPX domain protein that transports Pi into the vacuole in Arabidopsis. The vpt1 mutant plants were stunted and consistently retained less Pi than wild type plants, especially when grown in medium containing high levels of Pi. In seedlings, VPT1 was expressed primarily in younger tissues under normal conditions, but was strongly induced by high-Pi conditions in older tissues, suggesting that VPT1 functions in Pi storage in young tissues and in detoxification of high Pi in older tissues. As a result, disruption of VPT1 rendered plants hypersensitive to both low-Pi and high-Pi conditions, reducing the adaptability of plants to changing Pi availability. Patch-clamp analysis of isolated vacuoles showed that the Pi influx current was severely reduced in vpt1 compared with wild type plants. When ectopically expressed in Nicotiana benthamiana mesophyll cells, VPT1 mediates vacuolar influx of anions, including Pi, SO42−, NO3−, Cl−, and malate with Pi as that preferred anion. The VPT1-mediated Pi current amplitude was dependent on cytosolic phosphate concentration. Single-channel analysis showed that the open probability of VPT1 was increased with the increase in transtonoplast potential. We conclude that VPT1 is a transporter responsible for vacuolar Pi storage and is essential for Pi adaptation in Arabidopsis.

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CFTR in K562 human leukemic cells

AJP Cell Physiology ◽

10.1152/ajpcell.00320.2002 ◽

2003 ◽

Vol 285 (2) ◽

pp. C480-C488 ◽

Cited By ~ 7

Author(s):

Yanina A. Assef ◽

Alicia E. Damiano ◽

Elsa Zotta ◽

Cristina Ibarra ◽

Basilio A. Kotsias

Keyword(s):

Single Channel ◽

Functional Characterization ◽

Leukemic Cells ◽

Cftr Gene ◽

Human Leukemia ◽

Selectivity Ratio ◽

Open Probability ◽

Selective Channel ◽

Human Leukemic Cells

In this study, the expression and functional characterization of CFTR (cystic fibrosis transmembrane regulator) was determined in K562 chronic human leukemia cells. Expression of the CFTR gene product was determined by RT-PCR and confirmed by immunohistochemistry and Western blot analysis. Functional characterization of CFTR Cl- channel activity was conducted with patch-clamp techniques. Forskolin, an adenylyl cyclase activator, induced an anion-selective channel with a linear current-voltage relationship and a single-channel conductance of 11 pS. This cAMP-activated channel had a Pgluconate/PCl or PF/PCl perm-selectivity ratio of 0.35 and 0.30, respectively, and was inhibited by the CFTR blocker glibenclamide and the anti-CFTR antibody MAb 13-1, when added to the cytoplasmatic side of the patch. Glibenclamide decreased the open probability increasing the frequency of open-to-closed transitions. Addition of 200 μM DIDS caused an irreversible block of the channels when added to the cytosolic side of inside-out patches. These and other observations indicate a widespread distribution of CFTR gene expression and suggest that this channel protein may function in most human cells to help maintain cellular homeostasis.

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Elementary mechanisms of calmodulin regulation of NaV1.5 producing divergent arrhythmogenic phenotypes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2025085118 ◽

2021 ◽

Vol 118 (21) ◽

pp. e2025085118

Author(s):

Po Wei Kang ◽

Nourdine Chakouri ◽

Johanna Diaz ◽

Gordon F. Tomaselli ◽

David T. Yue ◽

...

Keyword(s):

Single Channel ◽

Loss Of Function ◽

Na Current ◽

Open Probability ◽

Atomic Structures ◽

Single Channel Analysis ◽

Inactivation Mechanism ◽

Life Threatening ◽

Channel Analysis ◽

Cam Regulation

In cardiomyocytes, NaV1.5 channels mediate initiation and fast propagation of action potentials. The Ca2+-binding protein calmodulin (CaM) serves as a de facto subunit of NaV1.5. Genetic studies and atomic structures suggest that this interaction is pathophysiologically critical, as human mutations within the NaV1.5 carboxy-terminus that disrupt CaM binding are linked to distinct forms of life-threatening arrhythmias, including long QT syndrome 3, a “gain-of-function” defect, and Brugada syndrome, a “loss-of-function” phenotype. Yet, how a common disruption in CaM binding engenders divergent effects on NaV1.5 gating is not fully understood, though vital for elucidating arrhythmogenic mechanisms and for developing new therapies. Here, using extensive single-channel analysis, we find that the disruption of Ca2+-free CaM preassociation with NaV1.5 exerts two disparate effects: 1) a decrease in the peak open probability and 2) an increase in persistent NaV openings. Mechanistically, these effects arise from a CaM-dependent switch in the NaV inactivation mechanism. Specifically, CaM-bound channels preferentially inactivate from the open state, while those devoid of CaM exhibit enhanced closed-state inactivation. Further enriching this scheme, for certain mutant NaV1.5, local Ca2+ fluctuations elicit a rapid recruitment of CaM that reverses the increase in persistent Na current, a factor that may promote beat-to-beat variability in late Na current. In all, these findings identify the elementary mechanism of CaM regulation of NaV1.5 and, in so doing, unravel a noncanonical role for CaM in tuning ion channel gating. Furthermore, our results furnish an in-depth molecular framework for understanding complex arrhythmogenic phenotypes of NaV1.5 channelopathies.

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The β Subunit Increases the Ca2+ Sensitivity of Large Conductance Ca2+-activated Potassium Channels by Retaining the Gating in the Bursting States

The Journal of General Physiology ◽

10.1085/jgp.113.3.425 ◽

1999 ◽

Vol 113 (3) ◽

pp. 425-440 ◽

Cited By ~ 99

Author(s):

Crina M. Nimigean ◽

Karl L. Magleby

Keyword(s):

Single Channel ◽

Bk Channels ◽

Bk Channel ◽

Β Subunit ◽

Open Probability ◽

Α Subunit ◽

Closed Intervals ◽

Single Channel Analysis ◽

Channel Analysis ◽

The Mean

Coexpression of the β subunit (KV,Caβ) with the α subunit of mammalian large conductance Ca2+- activated K+ (BK) channels greatly increases the apparent Ca2+ sensitivity of the channel. Using single-channel analysis to investigate the mechanism for this increase, we found that the β subunit increased open probability (Po) by increasing burst duration 20–100-fold, while having little effect on the durations of the gaps (closed intervals) between bursts or on the numbers of detected open and closed states entered during gating. The effect of the β subunit was not equivalent to raising intracellular Ca2+ in the absence of the beta subunit, suggesting that the β subunit does not act by increasing all the Ca2+ binding rates proportionally. The β subunit also inhibited transitions to subconductance levels. It is the retention of the BK channel in the bursting states by the β subunit that increases the apparent Ca2+ sensitivity of the channel. In the presence of the β subunit, each burst of openings is greatly amplified in duration through increases in both the numbers of openings per burst and in the mean open times. Native BK channels from cultured rat skeletal muscle were found to have bursting kinetics similar to channels expressed from alpha subunits alone.

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Elementary Mechanisms Producing Facilitation of Cav2.1 (P/Q-type) Channels

The Journal of General Physiology ◽

10.1085/jgp.200709749 ◽

2007 ◽

Vol 129 (5) ◽

pp. 385-401 ◽

Cited By ~ 40

Author(s):

Dipayan Chaudhuri ◽

John B. Issa ◽

David T. Yue

Keyword(s):

Charge Carrier ◽

Single Channel ◽

Open Probability ◽

Activation Kinetics ◽

Appreciable Change ◽

Channel Modulation ◽

Dependent Inactivation ◽

Single Channel Analysis ◽

The Family ◽

Channel Analysis

The regulation of CaV2.1 (P/Q-type) channels by calmodulin (CaM) showcases the powerful Ca2+ decoding capabilities of CaM in complex with the family of CaV1-2 Ca2+ channels. Throughout this family, CaM does not simply exert a binary on/off regulatory effect; rather, Ca2+ binding to either the C- or N-terminal lobe of CaM alone can selectively trigger a distinct form of channel modulation. Additionally, Ca2+ binding to the C-terminal lobe triggers regulation that appears preferentially responsive to local Ca2+ influx through the channel to which CaM is attached (local Ca2+ preference), whereas Ca2+ binding to the N-terminal lobe triggers modulation that favors activation via Ca2+ entry through channels at a distance (global Ca2+ preference). CaV2.1 channels fully exemplify these features; Ca2+ binding to the C-terminal lobe induces Ca2+-dependent facilitation of opening (CDF), whereas the N-terminal lobe yields Ca2+-dependent inactivation of opening (CDI). In mitigation of these interesting indications, support for this local/global Ca2+ selectivity has been based upon indirect inferences from macroscopic recordings of numerous channels. Nagging uncertainty has also remained as to whether CDF represents a relief of basal inhibition of channel open probability (Po) in the presence of external Ca2+, or an actual enhancement of Po over a normal baseline seen with Ba2+ as the charge carrier. To address these issues, we undertake the first extensive single-channel analysis of CaV2.1 channels with Ca2+ as charge carrier. A key outcome is that CDF persists at this level, while CDI is entirely lacking. This result directly upholds the local/global Ca2+ preference of the lobes of CaM, because only a local (but not global) Ca2+ signal is here present. Furthermore, direct single-channel determinations of Po and kinetic simulations demonstrate that CDF represents a genuine enhancement of open probability, without appreciable change of activation kinetics. This enhanced-opening mechanism suggests that the CDF evoked during action-potential trains would produce not only larger, but longer-lasting Ca2+ responses, an outcome with potential ramifications for short-term synaptic plasticity.

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