Isolation of an ion channel gene from <i>Arabidopsis thaliana</i> using the H5 signature sequence from voltage‐dependent K<sup>+</sup> channels

Ketchum, Karen A.; Slayman, Carolyn W.

doi:10.1016/0014-5793(95)01417-9

Cited by 89 publications

(59 citation statements)

References 40 publications

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“…Screening cDNA libraries using heterologous probes for transporters has similarly met with little success. However, the cloning of AKT2 / AKT3 using a conserved sequence of the K+-channel H5 pore domain is an example in which this approach was successful (Ketchum and Slayman, 1996).…”

Section: Expression Cloning and Heterologous Expressionmentioning

confidence: 99%

Patch-Clamping and Other Molecular Approaches for the Study of Plasma Membrane Transporters Demystified

Ward¹

1997

Plant Physiology

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As the boundary between the inside and the outside of the cell, the plasma membrane is one of the most important structures that cells use to regulate their interna1 composition and activities. Proteins embedded in the plasma membrane control which solutes are accumulated and which are excluded; their activity creates gradients of solutes across the membrane. This process is important for many aspects of plant growth and development, including cell expansion, mineral nutrition, long-distance transport of assimilated carbon and nitrogen, and cellular and whole-plant responses to environmental signals.Electrophysiology is the study of the transport of charged solutes across membranes and the proteins that carry out this transport. Electrophysiological studies have been performed on plants for at least 50 years. In the last 15 years the highest-resolution electrophysiological method, the patch-clamp technique, has been used extensively in plants. As a result, many plasma membrane transport activities, especially ion channel activities, have been characterized in detail. These activities can now be linked to structural information of proteins and, as a result, we can begin to understand the mechanisms of transport at the molecular level.Neither traditional genetic approaches nor Iibrary screening using heterologous probes has been useful for obtaining transport protein genes. A unique set of molecular tools, including expression cloning in yeast, heterologous expression in Xenopus laevis oocytes, reverse genetics to obtain knockout mutants, and high-resolution electrophysiological assays, is being used to begin to create a full description of transport protein structure, function, expression pattern, targeting, and regulation.This Update provides a practical guide for nonelectrophysiologists to understand the results from patch-clamp experiments. This is important because the patch-clamp technique will have additional applications in the future as knockout mutants and heterologous expression become more common. Furthermore, patch-clamp results are difficult to understand for many scientists who have not had personal experience with the technique. The current molecular approaches mentioned above, which are aiding in the rapid progression of the cloning of cDNAs of transport proteins, are also presented here to provide a glimpse into the cutting edge of electrophysiology, where mutant trans-* E-rnail wardauni-tuebingen.de; fax 049-07071-29-3287. porters are selected for desired characteristics on a functional basis and where knockout/ replacement studies will finally be possible in plants.

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Section: Expression Cloning and Heterologous Expressionmentioning

confidence: 99%

Patch-Clamping and Other Molecular Approaches for the Study of Plasma Membrane Transporters Demystified

Ward¹

1997

Plant Physiology

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“…Like Shaker ␣-subunits, cnggated cation channels cloned to date from animal systems have six membrane-spanning regions and a P (pore) region, with intracellular hydrophilic N and C termini (Zagotta and Siegelbaum, 1996). The pore region of (both animal and plant) K ϩ -selective voltage-gated channels retains a highly conserved signature pore sequence that determines K ϩ selectivity (Heginbotham et al, 1994;Ketchum and Slayman, 1996). The pore region of cng-gated channels (which, as noted above, do not display K ϩ selectivity) retains some but not all of the K ϩ channel signature pore sequence.…”

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confidence: 95%

Cloning and First Functional Characterization of a Plant Cyclic Nucleotide-Gated Cation Channel

et al. 1999

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“…AKT2 and AKT3 are two proteins encoded by the same gene (At4 g22200); AKT3 (Ketchum and Slayman, 1996;Marten et al, 1999;Hoth et al, 2001) represents a truncated version of AKT2 (Cao et al, 1995;Lacombe et al, 2000b) characterized by a 15-amino acid shorter cytoplasmic N terminus. The presence of one or both of these proteins in planta has not yet been determined.…”

Section: Introductionmentioning

confidence: 99%

Outer Pore Residues Control the H⁺ and K⁺ Sensitivity of the Arabidopsis Potassium Channel AKT3

et al. 2002

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The Arabidopsis phloem channel AKT3 is the founder of a subfamily of shaker -like plant potassium channels characterized by weak rectification, Ca 2 ؉ block, proton inhibition, and, as shown in this study, K ؉ sensitivity. In contrast to inward-rectifying, acid-activated K ؉ channels of the KAT1 family, extracellular acidification decreases AKT3 currents at the macroscopic and single-channel levels. Here, we show that two distinct sites within the outer mouth of the K ؉ -conducting pore provide the molecular basis for the pH sensitivity of this phloem channel. After generation of mutant channels and functional expression in Xenopus oocytes, we identified the His residue His-228, which is proximal to the K ؉ selectivity filter (GYGD) and the distal Ser residue Ser-271, to be involved in proton susceptibility. Mutations of these sites, H228D and S271E, drastically reduced the H ؉ and K ؉ sensitivity of AKT3. Although in K ؉ -free bath solutions outward K ؉ currents were abolished completely in wild-type AKT3, S271E as well as the AKT3-HDSE double mutant still mediated K ؉ efflux. We conclude that the pH-and K ؉ -dependent properties of the AKT3 channel involve residues in the outer mouth of the pore. Both properties, H ؉ and K ؉ sensitivity, allow the fine-tuning of the phloem channel and thus seem to represent important elements in the control of membrane potential and sugar loading.

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Isolation of an ion channel gene from Arabidopsis thaliana using the H5 signature sequence from voltage‐dependent K⁺ channels

Cited by 89 publications

References 40 publications

Patch-Clamping and Other Molecular Approaches for the Study of Plasma Membrane Transporters Demystified

Patch-Clamping and Other Molecular Approaches for the Study of Plasma Membrane Transporters Demystified

Cloning and First Functional Characterization of a Plant Cyclic Nucleotide-Gated Cation Channel

Outer Pore Residues Control the H⁺ and K⁺ Sensitivity of the Arabidopsis Potassium Channel AKT3

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Isolation of an ion channel gene from Arabidopsis thaliana using the H5 signature sequence from voltage‐dependent K+ channels

Cited by 89 publications

References 40 publications

Patch-Clamping and Other Molecular Approaches for the Study of Plasma Membrane Transporters Demystified

Patch-Clamping and Other Molecular Approaches for the Study of Plasma Membrane Transporters Demystified

Cloning and First Functional Characterization of a Plant Cyclic Nucleotide-Gated Cation Channel

Outer Pore Residues Control the H+ and K+ Sensitivity of the Arabidopsis Potassium Channel AKT3

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Isolation of an ion channel gene from Arabidopsis thaliana using the H5 signature sequence from voltage‐dependent K⁺ channels

Outer Pore Residues Control the H⁺ and K⁺ Sensitivity of the Arabidopsis Potassium Channel AKT3