Because of the large size and modest selectivity of the connexin hemichannel aqueous pore, hemichannel opening must be highly regulated to maintain cell viability. At normal resting potentials, this regulation is achieved predominantly by the physiological extracellular Ca2+ concentration, which drastically reduces hemichannel activity. Here, we characterize the Ca2+ regulation of channels formed by wild-type human connexin26 (hCx26) and its human mutations, D50N/Y, that cause aberrant hemichannel opening and result in deafness and skin disorders. We found that in hCx26 wild-type channels, deactivation kinetics are accelerated as a function of Ca2+ concentration, indicating that Ca2+ facilitates transition to, and stabilizes, the closed state of the hemichannels. The D50N/Y mutant hemichannels show lower apparent affinities for Ca2+-induced closing than wild-type channels and have more rapid deactivation kinetics, which are Ca2+ insensitive. These results suggest that D50 plays a role in (a) stabilizing the open state in the absence of Ca2+, and (b) facilitating closing and stabilization of the closed state in the presence of Ca2+. To explore the role of a negatively charged residue at position 50 in regulation by Ca2+, this position was substituted with a cysteine residue, which was then modified with a negatively charged methanethiosulfonate reagent, sodium (2-sulfanoethyl) methanethiosulfonate (MTSES)−. D50C mutant hemichannels display properties similar to those of D50N/Y mutants. Recovery of the negative charge with chemical modification by MTSES− restores the wild-type Ca2+ regulation of the channels. These results confirm the essential role of a negative charge at position 50 for Ca2+ regulation. Additionally, charge-swapping mutagenesis studies suggest involvement of a salt bridge interaction between D50 and K61 in the adjacent connexin subunit in stabilizing the open state in low extracellular Ca2+. Mutant cycle analysis supports a Ca2+-sensitive interaction between these two residues in the open state of the channel. We propose that disruption of this interaction by extracellular Ca2+ destabilizes the open state and facilitates hemichannel closing. Our data provide a mechanistic understanding of how mutations at position 50 that cause human diseases are linked to dysfunction of hemichannel gating by external Ca2+.

Aberrant opening of nonjunctional connexin hemichannels at the plasma membrane is associated with many diseases, including ischemia and muscular dystrophy. Proper control of hemichannel opening is essential to maintain cell viability and is achieved by physiological levels of extracellular Ca 2+ , which drastically reduce hemichannel activity. Here we examined the role of conserved charged residues that form electrostatic networks near the extracellular entrance of the connexin pore, a region thought to be involved in gating rearrangements of hemichannels. Molecular dynamics simulations indicate discrete sites for Ca 2+ interaction and consequent disruption of salt bridges in the open hemichannels. Experimentally, we found that disruption of these salt bridges by mutations facilitates hemichannel closing. Two negatively charged residues in these networks are putative Ca 2+ binding sites, forming a Ca 2+ -gating ring near the extracellular entrance of the pore. Accessibility studies showed that this Ca 2+ -bound gating ring does not prevent access of ions or small molecules to positions deeper into the pore, indicating that the physical gate is below the Ca 2+ -gating ring. We conclude that intra-and intersubunit electrostatic networks at the extracellular entrance of the hemichannel pore play critical roles in hemichannel gating reactions and are tightly controlled by extracellular Ca 2+ . Our findings provide a general mechanism for Ca 2+ gating among different connexin hemichannel isoforms.connexin | hemichannels | gating

Control of plasma membrane connexin hemichannel opening is indispensable, and is achieved by physiological extracellular divalent ion concentrations. Here, we explore the differences between regulation by Ca(2+) and Mg(2+) of human connexin26 (hCx26) hemichannels and the role of a specific interaction in regulation by Ca (2+). To effect hemichannel closure, the apparent affinity of Ca(2+) (0.33 mM) is higher than for Mg(2+) (1.8 mM). Hemichannel closure is accelerated by physiological Ca(2+) concentrations, but non-physiological concentrations of extracellular Mg(2+) are required for this effect. Our recent report provided evidence that extracellular Ca(2+) facilitates hCx26 hemichannel closing by disrupting a salt bridge interaction between positions D50 and K61 that stabilizes the open state. New evidence from mutant cycle analysis indicates that D50 also interacts with Q48. We find that the D50-Q48 interaction contributes to stabilization of the open state, but that it is relatively insensitive to disruption by extracellular Ca(2+) compared with the D50-K61 interaction.

Large-pore channels permeable to small molecules such as ATP, in addition to atomic ions, are emerging as important regulators in health and disease. Nonetheless, their mechanisms of molecular permeation and selectivity remain mostly unexplored. Combining fluorescence microscopy and electrophysiology, we developed a novel technique that allows kinetic analysis of molecular permeation through connexin and CALHM1 channels in Xenopus oocytes rendered translucent. Using this methodology, we found that (1) molecular flux through these channels saturates at low micromolar concentrations, (2) kinetic parameters of molecular transport are sensitive to modulators of channel gating, (3) molecular transport and ionic currents can be differentially affected by mutation and gating, and (4) N-terminal regions of these channels control transport kinetics and permselectivity. Our methodology allows analysis of how human disease–causing mutations affect kinetic properties and permselectivity of molecular signaling and enables the study of molecular mechanisms, including selectivity and saturability, of molecular transport in other large-pore channels.

The cytosolic reducing agent glutathione can reverse thiol modification of cysteine residues inside the pores of connexins and other channels permeable to large molecules.

We identified CALHM1 as a pore-forming subunit of a plasma membrane ion channel with weak ion selectivity and unique coupled allosteric gating regulation by voltage and extracellular Ca 2þ (Ca 2þ o) (PNAS 109: E1963 (2012)). CALHM1 is expressed in mouse cortical neurons where it accounts for low [Ca 2þ ] o-enhanced conductance and action potential firing. We recently determined that a CALHM1 channel is a hexamer with an estimated effective pore diameter~14Å .Extracellular adenosine 5'-triphosphate (ATP) plays critical roles in physiological and signal transduction processes. We examined whether ATP can permeate CALHM1 channels. Reducing [Ca 2þ ] o to activate CALHM1 induced ATP release from hCALHM1-expressing HeLa and COS-1 cells, and Xenopus oocytes. Neither CALHM1 expression nor lowering [Ca 2þ ] o caused cell damage. Involvement of other possible mechanisms was ruled out because ATP release was unaffected by Brefeldin A (vesicular release), DCPIB (volume-sensitive Cl À channels), A438079 (P2X7 receptors), heptanol and carbenoxolone (connexins and pannexins). In contrast, ruthenium red (RuR), which inhibits CALHM1 currents, abolished low [Ca 2þ ] o-evoked ATP release. Thus, CALHM1 expression induces a novel ATP permeability. Ca 2þ o inhibited ATP release with IC 50 = 495 mM and Hill coefficient of 1.9, kinetic properties similar to those of its gating regulation. Membrane depolarization activates CALHM1 channels in normal [Ca 2þ ] o. hCALHM1-expressing but not mocktransfected cells released ATP in response to high [K þ ] o-induced depolarization in normal [Ca 2þ ] o , which was inhibited by RuR but not by connexin and pannexin-1 blockers. Thus, regulation of ATP release is correlated with the gating properties of CALHM1 channels, indicating that the CALHM1 channel is the conduit for ATP release. These results demonstrate that CALHM1 is a voltage-gated ATP release channel that may contribute to ATP release in physiological and pathological conditions.

j = 0. The macroscopic G j showed substantial reduction (by 82% at V j = 100mV) when depolarizing Cx50expressing cell, whereas a moderate reduction (by 61% at V j = À100mV) when hyperpolarizing Cx50-expressing cell. This observation is in agreement with previous finding that Cx50 hemichannel gates when the cell is depolarized. Single Cx50/Cx50Cx36N heterotypic channel displayed a main conductance of 120pS at low V j pulses (5 20mV), but the conductance rectified with increasing V j pulses. Cx50/Cx50N9R heterotypic GJ channel also exhibited asymmetrical V jgating properties at macroscopic level, and single channel recordings indicated a main conductance of~150pS at V j = 20mV. These results demonstrate that the V j -gating properties and unitary conductance of Cx50 hemichannels were evidently modified when docking with Cx50-Cx36N or Cx50N9R mutants. These novel properties of heterotypic channels provide insights into the V j -gating mechanisms of Cx50 GJ channels.

Detection sensitivity from the ultraviolet to the near infrared spectral region is a key parameter to meet today's demand for handling smallest analyte amounts and short measurement times in the optical evaluation of miscellaneous samples. The introduction of single photon counting based data acquisition has proven to yield a major sensitivity increase and very high dynamic range-it is the ideal method for measuring weak luminescence. We present the hardware and handling optimization of a state of the art spectrometer for steady-state and time-resolved fluorescence measurements. The high sensitivity of the spectrometer was shown by measurements of popular fluorescent dyes as well as the Raman spectrum of water under well defined and reproducible conditions. The achieved sensitivity allows us to quantify singlet oxygen generation and to characterize the singlet oxygen phosphorescence decay, a prerequisite when studying photosensitisers like porphyrins and phthalocyanines used for example in photodynamic therapy (PDT). Moreover, with the help of an integrating sphere fluorescence quantum yields of low fluorescent samples like Ru(bpy)3 in water can be determined very precisely. The fibre connection of the spectrometer to a time-resolved fluorescence microscope (MicroTime100/200) was also realized. The combination of the advantages of both setups makes it e.g. possible to perform 2D-lifetime imaging with a freely tunable detection window for low luminescent samples even far into the near infrared region. The measurements with such a combination give not only the spectral and lifetime information of a luminescent sample but also the spatial information which is especially important for hetergeneous samples.