Dual-color quantum dot detection of a heterotetrameric potassium channel (hK<sub>Ca</sub>3.1)

Waschk, Daniel E.J.; Fabian, Anke; Budde, Thomas; Schwab, Albrecht

doi:10.1152/ajpcell.00053.2010

Cited by 10 publications

(10 citation statements)

References 34 publications

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“…The final proof for the expression of functional K Ca 3.1 channel proteins in the plasma membrane was obtained from patch clamp experiments. Using a KCl containing pipette solution we recorded an outwardly rectifying whole-cell current with the typical pharmacological properties of K Ca 3.1 channels ( n = 8; see Figure 1D [29]). Mean current density rises from 4.8 ± 1.0 pA/pF under control conditions to 24.9 ± 2.0 pA/pF in the presence of 50 μmol/l 1-EBIO.…”

Section: Resultsmentioning

confidence: 99%

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Ion channels in control of pancreatic stellate cell migration

Storck¹,

Hild²,

Schimmelpfennig³

et al. 2016

Oncotarget

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Pancreatic stellate cells (PSCs) play a critical role in the progression of pancreatic ductal adenocarcinoma (PDAC). Once activated, PSCs support proliferation and metastasis of carcinoma cells. PSCs even co-metastasise with carcinoma cells. This requires the ability of PSCs to migrate. In recent years, it has been established that almost all “hallmarks of cancer” such as proliferation or migration/invasion also rely on the expression and function of ion channels. So far, there is only very limited information about the function of ion channels in PSCs. Yet, there is growing evidence that ion channels in stromal cells also contribute to tumor progression. Here we investigated the function of KCa3.1 channels in PSCs. KCa3.1 channels are also found in many tumor cells of different origin. We revealed the functional expression of KCa3.1 channels by means of Western blot, immunofluorescence and patch clamp analysis. The impact of KCa3.1 channel activity on PSC function was determined with live-cell imaging and by measuring the intracellular Ca2+ concentration ([Ca2+]i). KCa3.1 channel blockade or knockout prevents the stimulation of PSC migration and chemotaxis by reducing the [Ca2+]i and calpain activity. KCa3.1 channels functionally cooperate with TRPC3 channels that are upregulated in PDAC stroma. Knockdown of TRPC3 channels largely abolishes the impact of KCa3.1 channels on PSC migration. In summary, our results clearly show that ion channels are crucial players in PSC physiology and pathophysiology.

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Section: Resultsmentioning

confidence: 99%

“…Functional expression of K Ca 3.1 channels was revealed by performing the patch clamp experiments using the whole cell configuration as described previously [29]. Experiments were performed at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Ion channels in control of pancreatic stellate cell migration

Storck¹,

Hild²,

Schimmelpfennig³

et al. 2016

Oncotarget

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“…Studies of a variety of channels and receptors have shown that subunit arrangement in the conformation of heterooligomers varies depending on which membrane protein is being analyzed. It has been proposed that individual proteins might assemble with a random arrangement that is dependent on their abundance (40)(41)(42)(43), with a random arrangement of preformed homomeric dimers (44), and some channels even assemble as heteromeric structures with fixed stoichiometry (45,46). With respect to aquaporins, it was reported that the AQP4 isoforms M1 and M23 seem to assemble as heterotetramers with a random arrangement (47).…”

Section: Discussionmentioning

confidence: 99%

Heteromerization of PIP aquaporins affects their intrinsic permeability

Yaneff

Sigaut

Marquez

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

101

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The plant aquaporin plasma membrane intrinsic proteins (PIP) subfamily represents one of the main gateways for water exchange at the plasma membrane (PM). A fraction of this subfamily, known as PIP1, does not reach the PM unless they are coexpressed with a PIP2 aquaporin. Although ubiquitous and abundantly expressed, the role and properties of PIP1 aquaporins have therefore remained masked. Here, we unravel how FaPIP1;1, a fruit-specific PIP1 aquaporin from Fragaria x ananassa, contributes to the modulation of membrane water permeability (P f ) and pH aquaporin regulation. Our approach was to combine an experimental and mathematical model design to test its activity without affecting its trafficking dynamics. We demonstrate that FaPIP1;1 has a high water channel activity when coexpressed as well as how PIP1-PIP2 affects gating sensitivity in terms of cytosolic acidification. PIP1-PIP2 random heterotetramerization not only allows FaPIP1;1 to arrive at the PM but also produces an enhancement of FaPIP2;1 activity. In this context, we propose that FaPIP1;1 is a key participant in the regulation of water movement across the membranes of cells expressing both aquaporins.T he plasma membrane (PM) is the first barrier that limits water exchange in plant cells. The rate of its water transport capacity is mainly associated with aquaporins. Among the seven aquaporin subfamilies described in the plant kingdom, only plasma membrane intrinsic proteins (PIP) and some members of the nodulin-26-like intrinsic proteins (NIP) and X intrinsic proteins (XIP) subfamilies have been shown to be preferentially localized at the PM (1, 2). Of these, PIP aquaporins appear to have a large role in controlling membrane water permeability, whereas NIP and XIP have been mainly described as solute transporters (2-4). Plant PIP aquaporins represent a conserved subfamily that has been historically divided into two subgroups due to their differences in primary structure, PIP1 and PIP2. Interestingly, PIP aquaporins compose ∼40% of the total aquaporin set, and the PIP1 and PIP2 ratio among different species is relatively constant (5-12). Fig. S1 shows the distribution of all aquaporin genes present in plants whose genome has been completely sequenced and analyzed. Antisense inhibition experiments on Arabidopsis thaliana PIP1 and PIP2 have suggested that the two subgroups of aquaporins contribute to root or leaf hydraulic conductivity in the same way (13). In several plant species, members of the PIP1 and PIP2 subgroups were shown to be coexpressed in the same cell type (14-17).Although PIP1 are as ubiquitous as PIP2, the functional properties of each type of channel are different. PIP2 are very well described as a homotetramer with high water transport activity (18, 19) and a gating mechanism unequivocally associated with specific and conserved amino acid motifs triggered by cytosolic acidification (20-22), phosphorylation (23, 24), or divalent cation concentration (22). In contrast, PIP1 have shown complex heterogeneity in water and solute transpor...

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“…It is thus possible that the drastic changes in activation time resulting from charge perturbation at positions 362 and 363 of KCa3.1 arise from R362 and E363 interacting with KCa3.1 constituents other than CaMBD2A and CaMBD2B, or auxiliary proteins yet to be identified. Of interest, dual-color quantum dot measurements have led to the conclusion that KCa3.1 channels are assembled from two homomeric dimers and not from four independent subunits (Waschk et al, 2011). As the structural model presented in Fig.…”

Section: Discussionmentioning

confidence: 99%

Contribution of the KCa3.1 channel–calmodulin interactions to the regulation of the KCa3.1 gating process

Morales

Garneau

Klein

et al. 2013

Journal of General Physiology

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The Ca2+-activated potassium channel of intermediate conductance, KCa3.1, is now emerging as a therapeutic target for a large variety of health disorders. The Ca2+ sensitivity of KCa3.1 is conferred by the Ca2+-binding protein calmodulin (CaM), with the CaM C-lobe constitutively bound to an intracellular domain of the channel C terminus. It was proposed on the basis of the crystal structure obtained for the C-terminal region of the rat KCa2.2 channel (rSK2) with CaM that the binding of Ca2+ to the CaM N-lobe results in CaM interlocking the C-terminal regions of two adjacent KCa3.1 subunits, leading to the formation of a dimeric structure. A study was thus undertaken to identify residues of the CaM N-lobe–KCa3.1 complex that either contribute to the channel activation process or control the channel open probability at saturating Ca2+ (Pomax). A structural homology model of the KCa3.1–CaM complex was first generated using as template the crystal structure of the C-terminal region of the rat KCa2.2 channel with CaM. This model was confirmed by cross-bridging residues R362 of KCa3.1 and K75 of CaM. Patch-clamp experiments were next performed, demonstrating that the solvation energy of the residue at position 367 in KCa3.1 is a key determinant to the channel Pomax and deactivation time toff. Mutations of residues M368 and Q364 predicted to form anchoring points for CaM binding to KCa3.1 had little impact on either toff or Pomax. Finally, our results show that channel activation depends on electrostatic interactions involving the charged residues R362 and E363, added to a nonpolar energy contribution coming from M368. We conclude that electrostatic interactions involving residues R362 and E363 and hydrophobic effects at M368 play a prominent role in KCa3.1 activation, whereas hydrophobic interactions at S367 are determinant to the stability of the CaM–KCa3.1 complex throughout gating.

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Dual-color quantum dot detection of a heterotetrameric potassium channel (hK_Ca3.1)

Abstract: Waschk DE, Fabian A, Budde T, Schwab A. Dual-color quantum dot detection of a heterotetrameric potassium channel (hKCa3.1). Am

Cited by 10 publications

References 34 publications

Ion channels in control of pancreatic stellate cell migration

Ion channels in control of pancreatic stellate cell migration

Heteromerization of PIP aquaporins affects their intrinsic permeability

Contribution of the KCa3.1 channel–calmodulin interactions to the regulation of the KCa3.1 gating process

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