ObjectiveLow voltage-activated (LVA) calcium channels are crucial for regulating oscillatory behavior in several types of neurons and other excitable cells. LVA channels dysfunction has been implicated in epilepsy, neuropathic pain, cancer, among other diseases. Unlike for High Voltage-Activated (HVA) channels, voltage-dependence and kinetics of currents carried by recombinant LVA, i.e., CaV3 channels, are quite similar to those observed in native currents. Therefore, whether these channels are regulated by HVA auxiliary subunits, remain controversial. Here, we used the α1-subunits of CaV3.1, CaV3.2, and CaV3.3 channels, together with HVA auxiliary β-subunits to perform electrophysiological, confocal microscopy and immunoprecipitation experiments, in order to further explore this possibility.ResultsFunctional expression of CaV3 channels is up-regulated by all four β-subunits, although most consistent effects were observed with the β1b-subunit. The biophysical properties of CaV3 channels were not modified by any β-subunit. Furthermore, although β1b-subunits increased colocalization of GFP-tagged CaV3 channels and the plasma membrane of HEK-293 cells, western blots analysis revealed the absence of physical interaction between CaV3.3 and β1b-subunits as no co-immunoprecipitation was observed. These results provide solid evidence that the up-regulation of LVA channels in the presence of HVA-β1b subunit is not mediated by a high affinity interaction between both proteins.Electronic supplementary materialThe online version of this article (10.1186/s13104-018-3917-1) contains supplementary material, which is available to authorized users.
change in the pore size due to the intrinsic heterogeneity in the fluorescently labeled MscL pentamers where various stoichiometry of the donors and acceptors are present. Furthermore, we have found that sticking the channel to a surface alters the function of them. To get around these two problems, we performed single-molecule FRET experiments using an Alternating Laser EXcitation (ALEX) apparatus. By using alternating lasers of wavelengths 488 nm and 561 nm to excite directly the donors (Alexa488) and acceptors (Alexa568) present in single diffusing MscL molecules which are incorporated in 50 nm liposomes, both the distance-based FRET efficiency E and stoichiometrybased ratio S could be measured. Our single-molecule experiments show that the addition of an asymmetric lipid (LPC) to the liposomes opens the channels, consistent with the results from ensemble measurements.
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