Gap junction channel gating

AbstractOver the last two decades, the view of gap junction (GJ) channel gating has changed from one with GJs having a single transjunctional voltage-sensitive (Vj-sensitive) gating mechanism to one with each hemichannel of a formed GJ channel, as well as unapposed hemichannels, containing two, molecularly distinct gating mechanisms. These mechanisms are termed fast gating and slow or ‘loop’ gating. It appears that the fast gating mechanism is solely sensitive to Vj and induces fast gating transitions between the open state and a particular substate, termed the residual conductance state. The slow gating mechanism is also sensitive to Vj, but there is evidence that this gate may mediate gating by transmembrane voltage (Vm), intracellular Ca2+ and pH, chemical uncouplers and GJ channel opening during de novo channel formation. A distinguishing feature of the slow gate is that the gating transitions appear to be slow, consisting of a series of transient substates en route to opening and closing. Published reports suggest that both sensorial and gating elements of the fast gating mechanism are formed by transmembrane and cytoplamic components of connexins among which the N terminus is most essential and which determines gating polarity. We propose that the gating element of the slow gating mechanism is located closer to the central region of the channel pore and serves as a ‘common’ gate linked to several sensing elements that are responsive to different factors and located in different regions of the channel

Sanitation in challenging environments (SCE) project: mobilising sector engagement and innovation in Cambodia

A challenging environment is considered to be anywhere conventional sanitation solutions are not appropriate due to difficult geographical and/or geological conditions. In Cambodia, 4.14 million people (WSP, 2011) (~27% of Cambodia’s population) live in areas affected by flooding, high groundwater, floating, riverbank or coastal conditions impacting their ability to access appropriate and improved sanitation solutions at all times. Engineers Without Borders Australia, in collaboration with partner organisations in Cambodia, in 2014 initiated the Sanitation in Challenging Environments (SCE) Project to mobilise sector actors, build capacity of implementing partners and drive innovation to contribute to WASH sector program effectiveness to achieve sanitation access in these areas. This paper shares the activities undertaken so far and key learnings

Single-channel SCAM Identifies Pore-lining Residues in the First Extracellular Loop and First Transmembrane Domains of Cx46 Hemichannels

Gap junction (GJ) channels provide an important pathway for direct intercellular transmission of signaling molecules. Previously we showed that fixed negative charges in the first extracellular loop domain (E1) strongly influence charge selectivity, conductance, and rectification of channels and hemichannels formed of Cx46. Here, using excised patches containing Cx46 hemichannels, we applied the substituted cysteine accessibility method (SCAM) at the single channel level to residues in E1 to determine if they are pore-lining. We demonstrate residues D51, G46, and E43 at the amino end of E1 are accessible to modification in open hemichannels to positively and negatively charged methanethiosulfonate (MTS) reagents added to cytoplasmic or extracellular sides. Positional effects of modification along the length of the pore and opposing effects of oppositely charged modifying reagents on hemichannel conductance and rectification are consistent with placement in the channel pore and indicate a dominant electrostatic influence of the side chains of accessible residues on ion fluxes. Hemichannels modified by MTS-EA+, MTS-ET+, or MTS-ES− were refractory to further modification and effects of substitutions with positively charged residues that electrostatically mimicked those caused by modification with the positively charged MTS reagents were similar, indicating all six subunits were likely modified. The large reductions in conductance caused by MTS-ET+ were visible as stepwise reductions in single-channel current, indicative of reactions occurring at individual subunits. Extension of single-channel SCAM using MTS-ET+ into the first transmembrane domain, TM1, revealed continued accessibility at the extracellular end at A39 and L35. The topologically complementary region in TM3 showed no evidence of reactivity. Structural models show GJ channels in the extracellular gap to have continuous inner and outer walls of protein. If representative of open channels and hemichannels, these data indicate E1 as constituting a significant portion of this inner, pore-forming wall, and TM1 contributing as pore-lining in the extracellular portion of transmembrane span