Influence of the whole-cell patch-clamp configuration on electrophysiological properties of the voltage-dependent sodium current expressed in MDA-MB-231 breast cancer cells

Brugada syndrome (BrS) is a known cause of sudden cardiac death (SCD) characterized by abnormal electrocardiograms and fatal arrhythmias. The variants in KCND3 encoding the KV4.3 potassium-channel (the α-subunit of the Ito) have seldom been reported in BrS. This study aimed to identify novel KCND3 variants associated with BrS and elucidate BrS pathogenesis. High-depth targeted sequencing was performed and the electrophysiological properties of the variants were detected by whole-cell patch-clamp methods in a cultured-cell expressing system. The transcriptional levels of KV4.3 in different genotypes were studied by real-time PCR. Western blot was used to assess channel protein expression. A novel KCND3heterozygous variant, c.1292G>A (Arg431His, R431H), was found in the proband. Whole-cell patch-clamp results revealed a gain-of-function phenotype in the variant, with peak Ito current density increased and faster recovery from inactivation. The expression of mutant Kv4.3 membrane protein increased and the cytoplasmic protein decreased, demonstrating that the membrane/cytoplasm ratio was significantly different. In conclusion, a novel KCND3 heterozygous variant was associated with BrS. The increased Ito current explained the critical role of KCND3 in the pathogenesis of BrS. Genetic screening for KCND3 could be useful for understanding the pathogenesis of BrS and providing effective risk stratification in the clinic.

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Persistent Na+ conductance in medium-sized neostriatal neurons: characterization using infrared videomicroscopy and whole cell patch-clamp recordings

Journal of Neurophysiology ◽

10.1152/jn.1995.74.3.1343 ◽

1995 ◽

Vol 74 (3) ◽

pp. 1343-1348 ◽

Cited By ~ 51

Author(s):

C. Cepeda ◽

S. H. Chandler ◽

L. W. Shumate ◽

M. S. Levine

Keyword(s):

Patch Clamp ◽

Sodium Current ◽

Repetitive Firing ◽

Channel Blockers ◽

Potential Generation ◽

Whole Cell ◽

Cell Patch Clamp ◽

Whole Cell Patch Clamp ◽

Outward Currents ◽

Ramp Voltage

1. In the present study we investigate the expression of a persistent Na+ conductance (INaP) in identified medium-sized neostriatal neurons. Nomarski optics and infrared videomicroscopy were used for cell visualization and identification in thick slices (350 microns). Current- and voltage-clamp recordings were obtained utilizing whole cell patch-clamp methodology. 2. Application of depolarizing ramp voltage commands from a holding potential of -70 mV induced a slow, noninactivating inward current that occurred before and independent of the rapidly inactivating sodium current that subserves action potential generation. INaP began to activate at potentials less negative than -70 mV and peaked at -34 +/- 1 (SE) mV. Its average peak amplitude was -100 +/- 17 pA. INaP was abolished by tetrodotoxin (TTX, 0.5-1 microM) or an Na(+)-free solution. In contrast, it was not affected by Ca2+ channel blockers. Depolarizing ramp commands also induced tetraethylammonium-sensitive outward currents. 3. Dopamine (DA) (20-100 microM) produced a significant reduction of INaP. 4. These results demonstrate the existence of a TTX-sensitive persistent Na+ conductance in medium-sized neostriatal neurons. This conductance is modulated by DA and could play a role in the generation of rhythmic oscillations and in supporting repetitive firing.

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Existence of voltage-dependent Ca2+ channels in vestibular dark cells: cytochemical and whole-cell patch-clamp studies

European Archives of Oto-Rhino-Laryngology ◽

10.1007/bf02905990 ◽

1997 ◽

Vol 254 (6) ◽

pp. 287-291 ◽

Cited By ~ 3

Author(s):

K. Imon ◽

T. Amano ◽

K. Ishihara ◽

M. Sasa ◽

K. Yajin

Keyword(s):

Patch Clamp ◽

Whole Cell ◽

Dark Cells ◽

Cell Patch Clamp ◽

Whole Cell Patch Clamp ◽

Voltage Dependent ◽

Vestibular Dark Cells ◽

Ca2 Channels

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Kvβ1.3 Reduces the Degree of Stereoselective Bupivacaine Block of Kv1.5 Channels

Anesthesiology ◽

10.1097/01.anes.0000282100.32923.5c ◽

2007 ◽

Vol 107 (4) ◽

pp. 641-651 ◽

Cited By ~ 14

Author(s):

Cristina Arias ◽

Miriam Guizy ◽

Miren David ◽

Stefanie Marzian ◽

Teresa González ◽

...

Keyword(s):

Patch Clamp ◽

Binding Site ◽

Voltage Clamp ◽

Xenopus Oocytes ◽

Time Dependent ◽

Hek293 Cells ◽

Whole Cell ◽

Cell Patch Clamp ◽

Whole Cell Patch Clamp ◽

Voltage Dependent

Background Kvbeta1.3 subunit modifies the gating and the pharmacology of Kv1.5 channels, decreasing their sensitivity to block induced by drugs, suggesting that Kvbeta1.3 competes with them for a binding site at Kv1.5 channels. Methods Currents generated by the activation of Kv1.5 and Kv1.5 + Kvbeta1.3 channels expressed in HEK293 cells and Xenopus oocytes were recorded by using whole cell patch clamp and voltage clamp techniques. Results Block of Kv1.5, but not that produced on Kv1.5 + Kvbeta1.3 channels, was voltage dependent. In both channels, bupivacaine block was time dependent. R(+)- and S(-)-bupivacaine blocked Kv1.5 with IC50 4.4 +/- 0.5 microM (n = 15) and 39.8 +/- 8.2 microM (n = 16; P < 0.05), respectively. These values increased fourfold for R(+)-bupivacaine (17.2 +/- 2.2 microM) and twofold for S(-)-bupivacaine (71.9 +/- 11.5 microM) in Kv1.5 + Kvbeta1.3 channels. Therefore, the degree of stereoselectivity (theta) decreased from 9 to 4 in the presence of Kvbeta1.3. The decrease in potency to block Kv1.5 + Kvbeta1.3 channels was the result of a less stable interaction between bupivacaine enantiomers and channels. Differences in stereoselectivity in each situation were due to a more favorable interaction between the channel and R(+)-bupivacaine. In the presence of Kvbeta1.3, stereoselectivity was abolished for V514A mutant channels (involved in bupivacaine binding but not in Kvbeta1.3 binding) but not for L510A (part of Kvbeta1.3 binding site). Conclusions The degree of stereoselective block of Kv1.5 decreases from 9 to 4 when Kvbeta1.3 is present. L510 is determinant for the modulation of bupivacaine block, because it is the only residue of the S6 segment that binds to both bupivacaine and Kvbeta1.3. These findings support an overlapping binding site for drugs and Kvbeta1.3.

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