Gap junction channels mediate intercellular signalling that is crucial in tissue development, homeostasis and pathologic states such as cardiac arrhythmias, cancer and trauma. To explore the mechanism by which Ca2+ blocks intercellular communication during tissue injury, we determined the X-ray crystal structures of the human Cx26 gap junction channel with and without bound Ca2+. The two structures were nearly identical, ruling out both a large-scale structural change and a local steric constriction of the pore. Ca2+ coordination sites reside at the interfaces between adjacent subunits, near the entrance to the extracellular gap, where local, side chain conformational rearrangements enable Ca2+chelation. Computational analysis revealed that Ca2+-binding generates a positive electrostatic barrier that substantially inhibits permeation of cations such as K+ into the pore. Our results provide structural evidence for a unique mechanism of channel regulation: ionic conduction block via an electrostatic barrier rather than steric occlusion of the channel pore.

The signaling specificity of five purified G protein ␤␥ dimers, ␤ 1 ␥ 2 , ␤ 2 ␥ 2 , ␤ 3 ␥ 2 , ␤ 4 ␥ 2 , and ␤ 5 ␥ 2 , was explored by reconstituting them with G s ␣ and receptors or effectors in the adenylyl cyclase cascade. The ability of the five ␤␥ dimers to support receptor-␣-␤␥ interactions was examined using membranes expressing the ␤ 1 -adrenergic or A2a adenosine receptors. These receptors discriminated among the defined heterotrimers based solely on the ␤ isoform. The ␤ 4 ␥ 2 dimer demonstrated the highest coupling efficiency to either receptor. The ␤ 5 ␥ 2 dimer coupled poorly to each receptor, with EC 50 values 40 -200-fold higher than those observed with ␤ 4 ␥ 2 . Strikingly, whereas the EC 50 of the ␤ 1 ␥ 2 dimer at the ␤ 1 -adrenergic receptor was similar to ␤ 4 ␥ 2 , its EC 50 was 20-fold higher at the A2a adenosine receptor. Inhibition of adenylyl cyclase type I (AC1) and stimulation of type II (AC2) by the ␤␥ dimers were measured. ␤␥ dimers containing G␤ 1-4 were able to stimulate AC2 similarly, and ␤ 5 ␥ 2 was much less potent. ␤ 1 ␥ 2 , ␤ 2 ␥ 2 , and ␤ 4 ␥ 2 inhibited AC1 equally; ␤ 3 ␥ 2 was 10-fold less effective, and ␤ 5 ␥ 2 had no effect. These data argue that the ␤ isoform in the ␤␥ dimer can determine the specificity of signaling at both receptors and effectors.Signal transduction involving heterotrimeric G proteins 1 is a universal mechanism for the integration of extracellular stimuli such as hormones, neurotransmitters, odorants, and light (1, 2). The components involved in this signaling cascade are diverse, including a large number of receptors, G protein ␣ and ␤␥ subunits and effectors. Even though the diversity of the proteins in this system could potentially account for the known specificity of signaling in differentiated cells, the mechanisms for determining specificity are not completely defined. The ␤-adrenergic receptor is one of the most well characterized seven transmembrane spanning receptors, and provides an excellent example of selective coupling to a particular ␣ subunit, G s . When activated, G s can stimulate all nine adenylyl cyclase isoforms (3, 4). The G protein ␤␥ dimer, when released after receptor activation, is also able to regulate adenylyl cyclase (5). However, the regulation of the various isoforms of adenylyl cyclase by the ␤␥ dimer is much more selective; apparently, only AC2, AC4 (6, 7), and AC7 (8) are stimulated by ␤␥, whereas the neuronal-specific AC1 (4) and possibly AC5 and AC6 are inhibited by the dimer (9). Moreover, there is evidence that AC2 does not respond well to dimers composed of certain ␤ and ␥ subunits (10) or to dimers containing the phosphorylated ␥ 12 subunit (11). Thus, to understand fully the regulation of adenylyl cyclase by a G s -coupled receptor, one needs to know which ␤␥ dimers are most likely to support receptor G protein coupling and the effects of ␤␥ dimers on the various isoforms of adenylyl cyclase.The number of functionally distinct ␤␥ dimers is potentially very large, with seven G protein ␤ isoforms (including two splice ...

Two-pore-domain K ؉ channels provide neuronal background currents that establish resting membrane potential and input resistance; their modulation provides a prevalent mechanism for regulating cellular excitability. The so-called TASK channel subunits (TASK-1 and TASK-3) are widely expressed, and they are robustly inhibited by receptors that signal through G␣q family proteins. Here, we manipulated G protein expression and membrane phosphatidylinositol 4,5-bisphosphate (PIP 2) levels in intact and cellfree systems to provide electrophysiological and biochemical evidence that inhibition of TASK channels by G␣q-linked receptors proceeds unabated in the absence of phospholipase C (PLC) activity, and instead involves association of activated G␣q subunits with the channels. Receptor-mediated inhibition of TASK channels was faster and less sensitive to a PLC␤1-ct minigene construct than inhibition of PIP2-sensitive Kir3.4(S143T) homomeric channels that is known to be dependent on PLC. TASK channels were strongly inhibited by constitutively active G␣q, even by a mutated version that is deficient in PLC activation. Receptor-mediated TASK channel inhibition required exogenous G␣q expression in fibroblasts derived from G␣q͞11 knockout mice, but proceeded unabated in a cell line in which PIP2 levels were reduced by regulated overexpression of a lipid phosphatase. Direct application of activated G␣q, but not other G protein subunits, inhibited TASK channels in excised patches, and constitutively active G␣q subunits were selectively coimmunoprecipitated with TASK channels. These data indicate that receptor-mediated TASK channel inhibition is independent of PIP2 depletion, and they suggest a mechanism whereby channel modulation by G␣q occurs through direct interaction with the ion channel or a closely associated intermediary.G protein ͉ KCNK ͉ neuromodulation ͉ phospholipase C ͉ TASK

It has been established that CaV3.2 T-type voltage-gated calcium channels (T-channels) play a key role in the sensitized (hyperexcitable) state of nociceptive sensory neurons (nociceptors) in response to hyperglycemia associated with diabetes, which in turn can be a basis for painful symptoms of peripheral diabetic neuropathy (PDN). Unfortunately, current treatment for painful PDN has been limited by nonspecific systemic drugs with significant side effects or potential for abuse. We studied in vitro and in vivo mechanisms of plasticity of CaV3.2 T-channel in a leptin-deficient (ob/ob) mouse model of PDN. We demonstrate that posttranslational glycosylation of specific extracellular asparagine residues in CaV3.2 channels accelerates current kinetics, increases current density, and augments channel membrane expression. Importantly, deglycosylation treatment with neuraminidase inhibits native T-currents in nociceptors and in so doing completely and selectively reverses hyperalgesia in diabetic ob/ob mice without altering baseline pain responses in healthy mice. Our study describes a new mechanism for the regulation of CaV3.2 activity and suggests that modulating the glycosylation state of T-channels in nociceptors may provide a way to suppress peripheral sensitization. Understanding the details of this regulatory pathway could facilitate the development of novel specific therapies for the treatment of painful PDN.

G protein-coupled inwardly rectifying potassium (GIRK) channels can be activated or inhibited by different classes of receptors, suggesting a role for G proteins in determining signaling specificity. Because G protein ␤␥ subunits containing either ␤1 or ␤2 with multiple G␥ subunits activate GIRK channels, we hypothesized that specificity might be imparted by ␤3, ␤4, or ␤5 subunits. We used a transfection assay in cell lines expressing GIRK channels to examine effects of dimers containing these G␤ subunits. Inwardly rectifying K ؉ currents were increased in cells expressing ␤3 or ␤4, with either ␥2 or ␥11. Purified, recombinant ␤3␥2 and ␤4␥2 bound directly to glutathione-S-transferase fusion proteins containing N-or C-terminal cytoplasmic domains of GIRK1 and GIRK4, indicating that ␤3 and ␤4, like ␤1, form dimers that bind to and activate GIRK channels. By contrast, ␤5-containing dimers inhibited GIRK channel currents. This inhibitory effect was obtained with either ␤5␥2 or ␤5␥11, was observed with either GIRK1,4 or GIRK1,2 channels, and was evident in the context of either basal or agonist-induced currents, both of which were mediated by endogenous G␤␥ subunits. In cotransfection assays, ␤5␥2 suppressed ␤1␥2-activated GIRK currents in a dose-dependent manner consistent with competitive inhibition. Moreover, we found that ␤5␥2 could bind to the same GIRK channel cytoplasmic domains as other, activating G␤␥ subunits. Thus, ␤5-containing dimers inhibit G␤␥-stimulated GIRK channels, perhaps by directly binding to the channels. This suggests that ␤5-containing dimers could act as competitive antagonists of other G␤␥ dimers on GIRK channels. P otassium channels that are active near resting membrane potentials are key determinants of cellular excitability. The G protein-coupled inwardly rectifying K ϩ (GIRK; Kir3.x) channels are particularly interesting in that they are differentially regulated by receptors that couple to different classes of heterotrimeric G proteins: GIRK channels are activated by receptors that couple to G␣i͞o and inhibited by receptors that couple to G␣q (1, 2). This dual up-and down-regulation of GIRK channels by different receptor classes has been described in atrial cells (3), aminergic brainstem neurons (4, 5), and enteric neurons of the peripheral nervous system (6).Mechanisms underlying inhibition of GIRK channels are not well understood. By contrast, the characteristics of receptormediated activation of GIRK channels have been worked out in detail. It is now clear that G␤␥ subunits liberated from G protein heterotrimers bind directly to GIRK channels to enhance channel activity (reviewed in refs. 1 and 2). This mechanism raises an interesting conundrum: If all G protein-coupled receptors release G␤␥ subunits when activated and all G␤␥ subunits tested to date activate GIRK channels (7), how is signaling specificity obtained such that different classes of receptor can activate or inhibit GIRK channels?One possibility is that specificity derives from associations of different receptors with par...