Cx26 (hCx26) channels seems to represent the channel with open gates. This structure, and molecular dynamics studies based on it, reveal that charged res- idues (D46, E47, R75, R184) at the extracellular entrance of the aqueous pore - a region thought to be involved in gating rearrangements - form an electrostatic network. We explored the role of these salt bridge interactions in gating using mutagenesis, kinetic analysis and chemical modifi cations. Substitution of neu- tral residues for D46 or E47, which would eliminate their participation in salt bridges, accelerate deactivation kinetics and moderately increase the apparent affi nity of Ca2to induce channel closing. These data support a role of these residues in stabilization of the open state. In addition, when D46 is substituted by a cysteine (D46C), modifi cation by MTSES to add a negative charge in- creases holding and tail currents. This suggests that a negative charge at this position is involved in stabilizing open hemichannels. In wild-type channels, following depolarizing pulses to 0 mV, peak tail currents increase as a function ofpulse duration,reaching maximumwithpulses of 40 sec. Strikingly,E47A/Q mutations showed peak tail currents that saturate more rapidly, at 15 sec, sug- gesting that this position also plays a key role in hemichannel activation. Thus far, our data suggest that intra- and inter-subunit electrostatic networks at the extracellular entrance of the hCx26 pore play critical roles in hemichannel gat- ing reactions. Support: R01GM099490. 3247-PosBoard B402 ExtracellularDivalentCationsRegulationofCx26andCx30 Hemichannels William Lopez, Yu Liu, Andrew L. Harris, Jorge E. Contreras. New Jersey Medical School-UMDNJ, Newark, NJ, USA. Due to the large size and modest selectivity of the aqueous pore, exacerbated opening of connexin hemichannels leads to loss of electrochemical gradients and of small cytoplasmic metabolites, causing cell death. Control of hemichan- nel opening is indispensable, and is achieved by extracellular divalent concen- trations, which drastically reduces hemichannel activity. Here, we explore the differences between extracellular Ca2and Mg2regulation in two relatives connexin, hCx26 and hCx30. Our standard protocol for assessment connexin hemichannel activation and deactivation with the two electro-voltage clamp technique is to examine the peak tail currents and their relaxation kinetics following a depolarizing pulse from ?80 mV to 0 mV. using this protocol, the peak tail currents increase with reduction of external divalents. We estimate the extracellular Ca2and Mg2 apparent affi nity for hCx26 hemichannels at values of 0.33 mM and 1.8 mM, respectively. hCx30 hemichannels showed slightly higher extracellular Ca2and Mg2 apparent affi nity with values of 0.17 mM and 1.0 mM, respectively. At physiological Ca2concentration (1.0 - 1.8 mM), both hCx26and hCx30hemichannels reach % 15% of the max- imal response, but at corresponding Mg2concentrations they reach R 50%. In addition, deactivation time constant at the tail currents are accelerated as a function of Ca2concentrations in hCx26 and hCx30 hemichannels; how- ever, only high extracellular Mg2concentrations ( 2.0 mM) are capable to accelerate deactivation kinetics in both connexin types. The holding currents at steady state are signifi cantly increased at physiological extracellular Mg2 concentrations (1.0 - 1.2 mM) in both hCx26 and hCx30 suggesting an increase in open hemichannels even at negative potentials. Our data support that, under physiological ionic conditions, Ca2, but not Mg2plays a major role stabiliz- ing and facilitating closing of Cx26 and Cx30 hemichannels. Support: R01GM099490 3248-PosBoard B403 The Effect of Arachidonic Acid on Junctional Conductance and Gating of Connexin 36 Gap Junction Channels and their Modulation by N-Alkanols Alina Marandykina1, Lina Rimkute 1, Nicola s Palacios-Prado2, Arvydas Skeberdis1, Feliksas Bukauskas2. 1Lithuanian University of Health Sciences Academy of Medicine, Kaunas, Lithuania, 2Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, New York, NY, USA. Arachidonic acid (AA)is oneof majorcomponents ofmembranephospholipids and is actively involved in the regulation of junctional conductance (gj). N- alkanols are well known inhibitors of gap junction (GJ) channels and have been shown to reduce neurological tremors. In this study, we examined function of Cx36 GJ channels, which are expressed in neurons and b-cells of pancreas, under control conditions and application of factors modulating con- centration of AA in the plasma membrane. We found that in HeLa cells expressing Cx36-EGFP, short carbon chain alkanols (SCCAs), such as penta- nol, hexanol and heptanol, increased gjby 3-fold. Conversely, long carbon chain alkanols (LCCAs), such as octanol, nonanoland decanol, uncoupled cells fully. We demonstrate that under control conditions only 0.003 of Cx36 GJ channels assembled in junction