Dataset Information

Mechanism of loss of Kv11.1 K+ current in mutant T421M-Kv11.1-expressing rat ventricular myocytes: interaction of trafficking and gating.

ABSTRACT: Type 2 long QT syndrome involves mutations in the human ether a-go-go-related gene (hERG or KCNH2). T421M, an S1 domain mutation in the Kv11.1 channel protein, was identified in a resuscitated patient. We assessed its biophysical, protein trafficking, and pharmacological mechanisms in adult rat ventricular myocytes.Isolated adult rat ventricular myocytes were infected with wild-type (WT)-Kv11.1- and T421M-Kv11.1-expressing adenovirus and analyzed with the use of patch clamp, Western blot, and confocal imaging techniques. Expression of WT-Kv11.1 or T421M-Kv11.1 produced peak tail current (I(Kv11.1)) of 8.78±1.18 and 1.91±0.22 pA/pF, respectively. Loss of mutant I(Kv11.1) resulted from (1) a partially trafficking-deficient channel protein with reduced cell surface expression and (2) altered channel gating with a positive shift in the voltage dependence of activation and altered kinetics of activation and deactivation. Coexpression of WT+T421M-Kv11.1 resulted in heterotetrameric channels that remained partially trafficking deficient with only a minimal increase in peak I(Kv11.1) density, whereas the voltage dependence of channel gating became WT-like. In the adult rat ventricular myocyte model, both WT-Kv11.1 and T421M-Kv11.1 channels responded to ?-adrenergic stimulation by increasing I(Kv11.1).The T421M-Kv11.1 mutation caused a loss of I(Kv11.1) through interactions of abnormal protein trafficking and channel gating. Furthermore, for coexpressed WT+T421M-Kv11.1 channels, different dominant-negative interactions govern protein trafficking and ion channel gating, and these are likely to be reflected in the clinical phenotype. Our results also show that WT and mutant Kv11.1 channels responded to ?-adrenergic stimulation.

SUBMITTER: Balijepalli SY

PROVIDER: S-EPMC3530382 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Mechanism of loss of Kv11.1 K+ current in mutant T421M-Kv11.1-expressing rat ventricular myocytes: interaction of trafficking and gating.

Balijepalli Sadguna Y SY Lim Evi E Concannon Sarah P SP Chew Chen L CL Holzem Kassandra E KE Tester David J DJ Ackerman Michael J MJ Delisle Brian P BP Balijepalli Ravi C RC January Craig T CT

Circulation 20121106 24

<h4>Background</h4>Type 2 long QT syndrome involves mutations in the human ether a-go-go-related gene (hERG or KCNH2). T421M, an S1 domain mutation in the Kv11.1 channel protein, was identified in a resuscitated patient. We assessed its biophysical, protein trafficking, and pharmacological mechanisms in adult rat ventricular myocytes.<h4>Methods and results</h4>Isolated adult rat ventricular myocytes were infected with wild-type (WT)-Kv11.1- and T421M-Kv11.1-expressing adenovirus and analyzed wi ...[more]

PMID: 23136156

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Project description:The effects of several phosphodiesterase (PDE) inhibitors on the L-type Ca current (I(Ca)) and intracellular cyclic AMP concentration ([cAMP]i) were examined in isolated rat ventricular myocytes. The presence of mRNA transcripts encoding for the different cardiac PDE subtypes was confirmed by RT-PCR. IBMX (100 microM), a broad-spectrum PDE inhibitor, increased basal I(Ca) by 120% and [cAMP]i by 70%, similarly to a saturating concentration of the beta-adrenoceptor agonist isoprenaline (1 microM). However, MIMX (1 microM), a PDE1 inhibitor, EHNA (10 microM), a PDE2 inhibitor, cilostamide (0.1 microM), a PDE3 inhibitor, or Ro20-1724 (0.1 microM), a PDE4 inhibitor, had no effect on basal I(Ca) and little stimulatory effects on [cAMP]i (20-30%). Each selective PDE inhibitor was then tested in the presence of another inhibitor to examine whether a concomitant inhibition of two PDE subtypes had any effect on I(Ca) or [cAMP]i. While all combinations tested significantly increased [cAMP]i (40-50%), only cilostamide (0.1 microM)+ Ro20-1724 (0.1 microM) produced a significant stimulation of I(Ca) (50%). Addition of EHNA (10 microM) to this mix increased I(Ca) to 110% and [cAMP]i to 70% above basal, i.e. to similar levels as obtained with IBMX (100 microM) or isoprenaline (1 microM). When tested on top of a sub-maximal concentration of isoprenaline (1 nM), which increased I(Ca) by (approximately 40% and had negligible effect on [cAMP]i, each selective PDE inhibitor induced a clear stimulation of [cAMP]i and an additional increase in I(Ca). Maximal effects on I(Ca) were approximately 8% for MIMX (3 microM), approximately 20% for EHNA (1-3 microM), approximately 30% for cilostamide (0.3-1 microM) and approximately 50% for Ro20-1724 (0.1 microM). Our results demonstrate that PDE1-4 subtypes regulate I(Ca) in rat ventricular myocytes. While PDE3 and PDE4 are the dominant PDE subtypes involved in the regulation of basal I(Ca), all four PDE subtypes determine the response of I(Ca) to a stimulus activating cyclic AMP production, with the rank order of potency PDE4>PDE3>PDE2>PDE1.

Dataset Information

Mechanism of loss of Kv11.1 K+ current in mutant T421M-Kv11.1-expressing rat ventricular myocytes: interaction of trafficking and gating.

Publications

Mechanism of loss of Kv11.1 K+ current in mutant T421M-Kv11.1-expressing rat ventricular myocytes: interaction of trafficking and gating.

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