Time-dependent expression of ryanodine receptors in sea urchin eggs, zygotes and early embryos

Summary In this work, the presence of calcium-dependent calcium channels and their receptors (RyR) has been investigated in Paracentrotus lividus eggs and early embryos, from unfertilized egg to four-blastomere stages. Electrophysiological recordings of RyR single-channel current fluctuations showed that RyRs are functional during the first developmental events with a maximum at zygote stage, c. 40 min after fertilization, corresponding to the first cleavage. The nature of vertebrate-like RyRs active at this stage was established by specific activation/blockade experiments.

Detecting the structural assembly pathway of human antimicrobial peptide pores at single-channel level

Elucidation of the assembly pathway of the human antimicrobial peptide channels by single-channel current recording.

Acute inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel by thyroid hormones involves multiple mechanisms

The chemical structures of the thyroid hormones triiodothyronine (T3) and thyroxine (T4) resemble those of small-molecules that inhibit the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. We therefore tested the acute effects of T3, T4 and reverse T3 (rT3) on recombinant wild-type human CFTR using the patch-clamp technique. When added directly to the intracellular solution bathing excised membrane patches, T3, T4, and rT3 (all tested at 50 μM) inhibited CFTR in several ways: they strongly reduced CFTR open probability by impeding channel opening; they moderately decreased single-channel current amplitude, and they promoted transitions to subconductance states. To investigate the mechanism of CFTR inhibition, we studied T3. T3 (50 μM) had multiple effects on CFTR gating kinetics, suggestive of both allosteric inhibition and open-channel blockade. Channel inhibition by T3 was weakly voltage dependent and stronger than the allosteric inhibitor genistein, but weaker than the open-channel blocker glibenclamide. Raising the intracellular ATP concentration abrogated T3 inhibition of CFTR gating, but not the reduction in single-channel current amplitude nor the transitions to subconductance states. The decrease in single-channel current amplitude was relieved by membrane depolarization, but not the transitions to subconductance states. We conclude that T3 has complex effects on CFTR consistent with both allosteric inhibition and open-channel blockade. Our results suggest that there are multiple allosteric mechanisms of CFTR inhibition, including interference with ATP-dependent channel gating and obstruction of conformational changes that gate the CFTR pore. CFTR inhibition by thyroid hormones has implications for the development of innovative small-molecule CFTR inhibitors.

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

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.

Tl+-induced μs Gating of Current Indicates Instability of the MaxiK Selectivity Filter as Caused by Ion/Pore Interaction

Patch clamp experiments on single MaxiK channels expressed in HEK293 cells were performed at high temporal resolution (50-kHz filter) in asymmetrical solutions containing 0, 25, 50, or 150 mM Tl+ on the luminal or cytosolic side with [K+] + [Tl+] = 150 mM and 150 mM K+ on the other side. Outward current in the presence of cytosolic Tl+ did not show fast gating behavior that was significantly different from that in the absence of Tl+. With luminal Tl+ and at membrane potentials more negative than −40 mV, the single-channel current showed a negative slope resistance concomitantly with a flickery block, resulting in an artificially reduced apparent single-channel current Iapp. The analysis of the amplitude histograms by β distributions enabled the estimation of the true single-channel current and the determination of the rate constants of a simple two-state O-C Markov model for the gating in the bursts. The voltage dependence of the gating ratio R = Itrue/Iapp = (kCO + kOC)/kCO could be described by exponential functions with different characteristic voltages above or below 50 mM Tl+. The true single-channel current Itrue decreased with Tl+ concentrations up to 50 mM and stayed constant thereafter. Different models were considered. The most likely ones related the exponential increase of the gating ratio to ion depletion at the luminal side of the selectivity filter, whereas the influence of [Tl+] on the characteristic voltage of these exponential functions and of the value of Itrue were determined by [Tl+] at the inner side of the selectivity filter or in the cavity.