K channels in T lymphocytes: a patch clamp study using monoclonal antibody adhesion

Matteson, D R; Deutsch, Carol

doi:10.1038/307468a0

Cited by 300 publications

(215 citation statements)

References 22 publications

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“…For data acquisition and analysis the pClamp10 software package (Molecular Devices, Sunnyvale, CA) were used. CD4 + T lymphocytes were selected for current recording by incubation with mouse anti-human CD4 antibodies (0.5 μg/10 6 cells, AMAC, Westbrook), followed by selective adhesion to petri dishes coated with goat anti-mouse IgG antibody (Biosource, Camarilo, CA), as described previously [39]. Standard whole-cell patch clamp techniques were used, as described previously [28].…”

Section: Electrophysiologymentioning

confidence: 99%

“…S5 and S6 segments with the pore loop between them form the pore domain (PD) of the channel which includes the ion conducting pore, whereas S1-S4 and the positively charged amino acids in S4 make up the voltage-sensing domain (VSD). Kv1.3, which belongs to the Shaker subfamily of Kv channels, is the predominant voltage-gated K + channel of human T lymphocytes [18,39].The main function of Kv1.3 channels during T cell activation is the maintenance of a permissive membrane potential (approximately equal to −50 mV) required for appropriate extracellular Ca 2+ entry and proper Ca 2+ signaling. By the use of Kv1.3-selective antagonists, lymphocyte proliferation and IL-2 production can be inhibited [14,47].…”

Section: Introductionmentioning

confidence: 99%

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7DHC-induced changes of Kv1.3 operation contributes to modified T cell function in Smith-Lemli-Opitz syndrome

Balajthy

Somodi

Pethő

et al. 2016

Pflugers Arch - Eur J Physiol

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In vitro manipulation of membrane sterol level affects the regulation of ion channels and consequently certain cellular functions; however, a comprehensive study that confirms the pathophysiological significance of these results is missing. The malfunction of 7-dehydrocholesterol (7DHC) reductase in Smith-Lemli-Opitz syndrome (SLOS) leads to the elevation of the 7-dehydrocholesterol level in the plasma membrane. T lymphocytes were isolated from SLOS patients to assess the effect of the in vivo altered membrane sterol composition on the operation of the voltage-gated Kv1.3 channel and the ion channel-dependent mitogenic responses. We found that the kinetic and equilibrium parameters of Kv1.3 activation changed in SLOS cells. Identical changes in Kv1.3 operation were observed when control/healthy T cells were loaded with 7DHC. Removal of the putative sterol binding sites on Kv1.3 resulted in a phenotype that was not influenced by the elevation in membrane sterol level. Functional assays exhibited impaired activation and proliferation rate of T cells probably partially due to the modified Kv1.3 operation. We concluded that the altered membrane sterol composition hindered the operation of Kv1.3 as well as the ion channel-controlled T cell functions.

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Section: Electrophysiologymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

7DHC-induced changes of Kv1.3 operation contributes to modified T cell function in Smith-Lemli-Opitz syndrome

Balajthy

Somodi

Pethő

et al. 2016

Pflugers Arch - Eur J Physiol

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“…They remained in tissue culture for -I wk following initial harvesting and were used for electrophysiological experiments throughout the culture period. Experiments were performed on cells rounded in shape and estimated at [20][21][22][23][24][25][26][27][28][29][30] Am in diameter. All experiments were carried out at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Immunoglobulin G-induced single ionic channels in human alveolar macrophage membranes.

Nelson¹,

Jacobs²,

Tang³

et al. 1985

J. Clin. Invest.

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While it is well known that the engagement of IgG Fc receptors on the macrophage surface triggers a number of cellular responses, including particle ingestion, secretion, and respiratory burst activity, the mechanism of signal transmission following ligand binding remains poorly understood. To acquire more data in this area, we studied the electrical properties of the macrophage membrane and its response to oligomeric immunoglobulin G (IgG) using the patch-clamp technique on human alveolar macrophages that were obtained by bronchoalveolar lavage and maintained in short-term tissue culture. The results showed that cell resting potentials, as determined from wholecell tight seal recordings, increased from -15 mV on the day of plating to -56 mV after the first day in culture and remained stable at this hyperpolarized level. Macrophages revealed an input resistance of 3.3 GO, independent of age in culture. Extracellular application of heat-aggregated human IgG to cells voltage-clamped at -70 mV resulted in peak inward currents of -470 pA. We identified an IgG-dependent, nonselective channel in both cell-attached and isolated membrane patches, with a unitary conductance of -350 pS and a predominant subconductance level of 235 pS in symmetrical NaCl solutions. Single channel open times were observed to be in the range of seconds and, in addition, were dependent upon membrane voltage. Channel opening involved transitions between a number of kinetic states and subconductance levels. Channel events recorded in cell-attached patches showed characteristic exponential relaxations, which implied a variation in membrane potential as a result of a single ion channel opening. These data suggest that the IgG-dependent nonselective cation channel that we have characterized may provide the link between Fc receptor engagement and subsequent cellular activation.

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“…The fact that inhibition of Kv1.3 in lymphocytes 26,27 is immuno-suppressive 28 motivated us to test Sa SMase C against human Kv1.3. We found that Sa SMase C eliminates well over half of the ionic current of Kv1.3 and of the gating current of its non-conducting W384F mutant 29 (Fig.…”

mentioning

confidence: 99%

Removal of phospho-head groups of membrane lipids immobilizes voltage sensors of K+ channels

Ramu

Lü

2008

Nature

170

161

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A fundamental question regarding the gating mechanism of voltage-activated K + (Kv) channels is how five positively charged voltage-sensing residues in the fourth transmembrane (TM) segment 1, 2 are energetically stabilized, as they operate in a low-dielectric cell membrane. The simplest solution would be to pair them with negative charges 3 . However, too few negatively charged channel residues are positioned for such a role 4,5 . Recent studies suggest that some of the channel's positively charged residues are exposed to cell membrane phospholipids and interact with their head groups [5][6][7][8][9] . A key question nonetheless remains: Is the phospho-head of membrane lipids necessary for proper function of the voltage sensor itself? Here, we find that a given type of Kv channel may interact with several species of phospholipid, and that enzymatic removal of their negatively charged phospho-head creates an insuperable energy barrier for the positively charged voltage sensor to move through the initial gating step(s), thus immobilizing it, and also raises the energy barrier for the downstream step(s).Kv2.1 channels, expressed in Xenopus oocytes, interact with sphingomyelin 8 present mainly in the outer leaf of plasma membranes. To investigate the importance of phospho-head groups of membrane lipids in Kv channel gating we employed bacterial sphingomyelinases C and D (SMases C and D) 10,11 . Both enzymes specifically hydrolyze sphingomyelin but in different ways (Fig. 1a): SMase D removes only choline and leaves the lipid ceramide-1-phosphate behind in the membrane whereas SMase C removes phosphocholine, leaving ceramide behind 12,13 . A comparison of the effects of these two enzymes on the channels will thus help reveal the functional significance of the phosphodiester group in voltage gating.SMase D of Corynebacterium pseudotuberculosis 11 shifts the conductance-voltage (G-V) relation of Kv2.1 by about -30 mV 8 (Fig. 1d), allowing channels to be activated at a negative voltage where they otherwise remain largely deactivated (Fig. 1c). The effect is maximal within 2 minutes and persists for at least 24 hours ( Fig. 1c, d; cells cannot regenerate sphingomyelin from ceramide-1-phosphate). It does not require direct exposure of channels to SMase D as it also occurred in Kv2.1-expressing oocytes that had been treated with SMase D and then thoroughly washed prior to injection with Kv2.1 cRNA (Fig. 1e).* Please address correspondence to: Dr. Zhe Lu, University of Pennsylvania, Department of Physiology, D302A Richards Building, 3700 Hamilton Walk, Philadelphia, PA 19104, Tel: 215-573-7711, FAX: 215-573-1940, zhelu@mail.med.upenn Additional exposure of such oocytes to SMase D, as expected, caused no further shift. Thus, SMase D acts through its lipase activity, not by direct binding to the channel. Our previous study 8 supports an electrostatic mechanism whereby removal of the positively charged choline favors the activated state of the positively charged voltage sensors.Unlike SMase D, SMase C removes the nega...

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K channels in T lymphocytes: a patch clamp study using monoclonal antibody adhesion

Cited by 300 publications

References 22 publications

7DHC-induced changes of Kv1.3 operation contributes to modified T cell function in Smith-Lemli-Opitz syndrome

7DHC-induced changes of Kv1.3 operation contributes to modified T cell function in Smith-Lemli-Opitz syndrome

Immunoglobulin G-induced single ionic channels in human alveolar macrophage membranes.

Removal of phospho-head groups of membrane lipids immobilizes voltage sensors of K+ channels

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