Heterologous Expression of Ion Channels in Mammalian Cell Lines

Mathie, Alistair; Veale, Emma L.; Holden, Robyn G

doi:10.1007/978-1-0716-0818-0_3

Cited by 8 publications

(7 citation statements)

References 17 publications

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“…This was significantly different [ p < 0.001, (95% CI = 111 to 412)] to cells expressing 500 ng GFP alone, ( Figures 2B, E ), which, when stimulated with the same voltage-protocol ( Figures 2B, F ), displayed small currents of 33 pA pF −1 ( n = 13) 95% CI = 28 to 37 ( Figure 2E ). Untransfected (or GFP alone transfected) tsA201 cells naturally express a small background K V component (see Figure 2B ), which can be blocked by TEA (10 mM), significantly reducing ( p < 0.0001, 95% CI: −34 to −17) the endogenous current to 7.4 pA pF −1 ( n = 4) 95% CI = 0.4 to 14 (data not shown, see also Mathie et al, 2021 ).…”

Section: Resultsmentioning

confidence: 84%

“…All individual expression plasmids were transiently co-expressed along with the green fluorescent reporter gene GFP at a concentration of 0.5 µg per well (0.25 µg per well for the toxin experiments to allow for adequate voltage control), using a modified calcium-phosphate protocol, as previously described ( Cunningham et al, 2019 ; Mathie et al, 2021 ) in a modified human embryonic kidney 293 cell line, tsA201 (European Collection of Authenticated Cell Cultures, Sigma-Aldrich, United Kingdom) prepared and maintained as previously described ( Veale et al, 2014a ; Cunningham et al, 2019 ; Mathie et al, 2021 ). For experiments where K V 2.1 or K V 2.1_P17T was co-expressed with the K V 9.3 subunit a ratio of 0.5:0.5 µg was transfected.…”

Section: Methodsmentioning

confidence: 99%

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A KCNB1 gain of function variant causes developmental delay and speech apraxia but not seizures

et al. 2022

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Objective: Numerous pathogenic variants in KCNB1, which encodes the voltage-gated potassium channel, KV2.1, are linked to developmental and epileptic encephalopathies and associated with loss-of-function, -regulation, and -expression of the channel. Here we describe a novel de novo variant (P17T) occurring in the KV2.1 channel that is associated with a gain-of-function (GoF), with altered steady-state inactivation and reduced sensitivity to the selective toxin, guanxitoxin-1E and is clinically associated with neurodevelopmental disorders, without seizures.Methods: The autosomal dominant variant was identified using whole exome sequencing (WES). The functional effects of the KCNB1 variant on the encoded KV2.1 channel were investigated using whole-cell patch-clamp recordings.Results: We identified a de novo missense variant in the coding region of the KCNB1 gene, c.49C>A which encodes a p.P17T mutation in the N-terminus of the voltage-gated, KV2.1 potassium channel. Electrophysiological studies measuring the impact of the variant on the functional properties of the channel, identified a gain of current, rightward shifts in the steady-state inactivation curve and reduced sensitivity to the blocker, guanxitoxin-1E.Interpretation: The clinical evaluation of this KCNB1 mutation describes a novel variant that is associated with global developmental delays, mild hypotonia and joint laxity, but without seizures. Most of the phenotypic features described are reported for other variants of the KCNB1 gene. However, the absence of early-onset epileptic disorders is a much less common occurrence. This lack of seizure activity may be because other variants reported have resulted in loss-of-function of the encoded KV2.1 potassium channel, whereas this variant causes a gain-of-function.

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Section: Resultsmentioning

confidence: 84%

Section: Methodsmentioning

confidence: 99%

A KCNB1 gain of function variant causes developmental delay and speech apraxia but not seizures

et al. 2022

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“…Most of the methods used here have been described previously [ 20 , 21 ] and a detailed explanation can be found in Ref. [ 22 ]. A summary of the methods used and any variations specific to this study are given below.…”

Section: Methodsmentioning

confidence: 99%

“…All WT and mutant constructs, including a green fluorescent protein (GFP)-expressing vector, were transiently transfected at a concentration of 0.5 μg, into tsA201 cells, using a modified calcium-phosphate transfection method [ 20 , 22 ].…”

Section: Methodsmentioning

confidence: 99%

Block of TREK and TRESK K2P channels by lamotrigine and two derivatives sipatrigine and CEN-092

Walsh

Leach

Veale

et al. 2021

Biochemistry and Biophysics Reports

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TREK and TRESK K2P channels are widely expressed in the nervous system, particularly in sensory neurons, where they regulate neuronal excitability. In this study, using whole-cell patch-clamp electrophysiology, we characterise the inhibitory effect of the anticonvulsant lamotrigine and two derivatives, sipatrigine and 3,5-diamino-6-(3,5-bistrifluoromethylphenyl)-1,2,4-triazine (CEN-092) on these channels. Sipatrigine was found to be a more effective inhibitor than lamotrigine of TREK-1, TREK-2 and TRESK channels. Sipatrigine was slightly more potent on TREK-1 channels (EC 50 = 16 μM) than TRESK (EC 50 = 34 μM) whereas lamotrigine was equally effective on TREK-1 and TRESK. Sipatrigine was less effective on a short isoform of TREK-2, suggesting the N terminus of the channel is important for both inhibition and subsequent over-recovery. Inhibition of TREK-1 and TREK-2 channels by sipatrigine was reduced by mutation of a leucine residue associated with the norfluoxetine binding site on these channels (L289A and L320A on TREK-1 and TREK-2, respectively) but these did not affect inhibition by lamotrigine. Inhibition of TRESK by sipatrigine and lamotrigine was attenuated by mutation of bulky phenylalanine residues (F145A and F352A) in the inner pore helix. However, phosphorylation mutations did not alter the effect of sipatrigine. CEN-092 was a more effective inhibitor of TRESK channels than TREK-1 channels. It is concluded that lamotrigine, sipatrigine and CEN-092 are all inhibitors of TREK and TRESK channels but do not greatly discriminate between them. The actions of these compounds may contribute to their current and potential use in the treatment of pain and depression.

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“…Most of the methods used here have been described previously ( Veale et al, 2014 ; Cunningham et al, 2019 ; Mathie et al, 2021a ) and will only be mentioned in brief below.…”

Section: Methodsmentioning

confidence: 99%

The Prostacyclin Analogue, Treprostinil, Used in the Treatment of Pulmonary Arterial Hypertension, is a Potent Antagonist of TREK-1 and TREK-2 Potassium Channels

2021

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Pulmonary arterial hypertension (PAH) is an aggressive vascular remodeling disease that carries a high morbidity and mortality rate. Treprostinil (Remodulin) is a stable prostacyclin analogue with potent vasodilatory and anti-proliferative activity, approved by the FDA and WHO as a treatment for PAH. A limitation of this therapy is the severe subcutaneous site pain and other forms of pain experienced by some patients, which can lead to significant non-compliance. TWIK-related potassium channels (TREK-1 and TREK-2) are highly expressed in sensory neurons, where they play a role in regulating sensory neuron excitability. Downregulation, inhibition or mutation of these channels leads to enhanced pain sensitivity. Using whole-cell patch-clamp electrophysiological recordings, we show, for the first time, that treprostinil is a potent antagonist of human TREK-1 and TREK-2 channels but not of TASK-1 channels. An increase in TASK-1 channel current was observed with prolonged incubation, consistent with its therapeutic role in PAH. To investigate treprostinil-induced inhibition of TREK, site-directed mutagenesis of a number of amino acids, identified as important for the action of other regulatory compounds, was carried out. We found that a gain of function mutation of TREK-1 (Y284A) attenuated treprostinil inhibition, while a selective activator of TREK channels, BL-1249, overcame the inhibitory effect of treprostinil. Our data suggests that subcutaneous site pain experienced during treprostinil therapy may result from inhibition of TREK channels near the injection site and that pre-activation of these channels prior to treatment has the potential to alleviate this nociceptive activity.

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Heterologous Expression of Ion Channels in Mammalian Cell Lines

Cited by 8 publications

References 17 publications

A KCNB1 gain of function variant causes developmental delay and speech apraxia but not seizures

A KCNB1 gain of function variant causes developmental delay and speech apraxia but not seizures

Block of TREK and TRESK K2P channels by lamotrigine and two derivatives sipatrigine and CEN-092

The Prostacyclin Analogue, Treprostinil, Used in the Treatment of Pulmonary Arterial Hypertension, is a Potent Antagonist of TREK-1 and TREK-2 Potassium Channels

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