Ca2+ block and flickering both contribute to the negative slope of the IV curve in BK channels

Abstract

Single-channel current–voltage (IV) curves of human large-conductance, voltage- and Ca2+-activated K+ (BK) channels are quite linear in 150 mM KCl. In the presence of Ca2+ and/or Mg2+, they show a negative slope conductance at high positive potentials. This is generally explained by a Ca2+/Mg2+ block as by Geng et al. (2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210955) in this issue. Here, we basically support this finding but add a refinement: the analysis of the open-channel noise by means of β distributions reveals what would be found if measurements were done with an amplifier of sufficient temporal resolution (10 MHz), namely that the block by 2.5 mM Ca2+ and 2.5 mM Mg2+ per se would only cause a saturating curve up to +160 mV. Further bending down requires the involvement of a second process related to flickering in the microsecond range. This flickering is hardly affected by the presence or absence of Ca2+/Mg2+. In contrast to the experiments reported here, previous experiments in BK channels (Schroeder and Hansen. 2007. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.200709802) showed saturating IV curves already in the absence of Ca2+/Mg2+. The reason for this discrepancy could not be identified so far. However, the flickering component was very similar in the old and new experiments, regardless of the occurrence of noncanonical IV curves.

Single-channel recordings of RyR1 at microsecond resolution in CMOS-suspended membranes

“…This in turn, would allow the measurement techniques here to be applied to ion channels with conductance of ∼450 pS. Although still a high conductance, there are a number of ion channels that could be studied, including the BK channel and bacterial homologs, such as MthK, or the largeconductance mechanosensitive channels (MscL) (37,38). Further reduction of the membrane size and improvements in the electronics have the potential to extend these techniques to lower conductance channels.…”

How to resolve microsecond current fluctuations in single ion channels: The power of beta distributions

“…2A and thus has to be determined by the analysis. 32,78 The viral K C channel Kcv NTS shows flickering at positive and negative membrane voltages. 79 At negative voltages, the resulting open-channel noise is extreme (arrow "g" in Fig.…”

“…Surprisingly, it was not possible to fit the amplitude histograms under the assumption that the singlepore current switched between fully open (I true ) and fully closed (I min D 0) as in the case of ion channels. 78,94,116 The best fit was obtained when blocking was assumed to switch between I min D 0.3 I true and I true . 80 This demonstrates that with sufficient data quality the lower level of the block (or channel closing) can be estimated from the fit in the same way as the true open-channel current.…”

“…For example, a typical bilayer experiment in our lab has a noise rms of 0.5 pA at a bandwidth of 1 kHz (unpublished data), whereas single-channel patch clamp experiments reach 1.5 pA noise at a much higher bandwidth of 20 kHz. 78 This has the consequence that the filter for the bilayer has to be set to values around 1-2 kHz in order to achieve an acceptable signal-to-noise ratio. Thus, some interesting properties in the time series of singlechannel traces are not detected by the low temporal resolution of traditional approaches like dwell-time analysis.…”

“…With and without Ca 2C , the flickery current reduction is of the same magnitude. 78 This indicates that 2 effects are responsible for the negative slope found in BK at high potentials.…”