Lymphedema is the clinical manifestation of defects in lymphatic structure or function. Mutations identified in genes regulating lymphatic development result in inherited lymphedema. No mutations have yet been identified in genes mediating lymphatic function that result in inherited lymphedema. Survey microarray studies comparing lymphatic and blood endothelial cells identified expression of several connexins in lymphatic endothelial cells. Additionally, gap junctions are implicated in maintaining lymphatic flow. By sequencing GJA1, GJA4, and GJC2 in a group of families with dominantly inherited lymphedema, we identified six probands with unique missense mutations in GJC2 (encoding connexin [Cx] 47). Two larger families cosegregate lymphedema and GJC2 mutation (LOD score = 6.5). We hypothesize that missense mutations in GJC2 alter gap junction function and disrupt lymphatic flow. Until now, GJC2 mutations were only thought to cause dysmyelination, with primary expression of Cx47 limited to the central nervous system. The identification of GJC2 mutations as a cause of primary lymphedema raises the possibility of novel gap-junction-modifying agents as potential therapy for some forms of lymphedema.

The understanding of neurotransmitter release at vertebrate synapses has been hampered by the paucity of preparations in which presynaptic ionic currents and postsynaptic responses can be monitored directly. We used cultured embryonic Xenopus neuromuscular junctions and simultaneous pre-and postsynaptic patch-clamp current-recording procedures to identify the major presynaptic conductances underlying the initiation of neurotransmitter release.Step depolarizations and action potential waveforms elicited Na and K currents along with Ca and Ca-activated K (K Ca ) currents. The onset of K Ca current preceded the peak of the action potential. The predominantly -CgTX GVIA-sensitive Ca current occurred primarily during the falling phase, but there was also significant Ca 2ϩ entry during the rising phase of the action potential. The postsynaptic current began a mean of 0.7 msec after the time of maximum rate of rise of the Ca current. -CgTX also blocked K Ca currents and transmitter release during an action potential, suggesting that Ca and K Ca channels are colocalized at presynaptic active zones. In double-ramp voltage-clamp experiments, K Ca channel activation is enhanced during the second ramp. The 1 msec time constant of decay of enhancement with increasing interpulse interval may reflect the time course of either the deactivation of K Ca channels or the diffusion/removal of Ca 2ϩ from sites of neurotransmitter release after an action potential. Key word: neuromuscular junction; nerve terminal; calcium channel; charybdotoxin; conotoxin; synaptic delayNeurotransmitter release from nerve terminals is triggered by the entry of Ca 2ϩ through voltage-gated Ca channels (Katz, 1969;Augustine et al., 1987). Our understanding of the relationship between the presynaptic ionic currents and release is based largely on studies of the squid giant synapse (Katz and Miledi, 1967;Llinás et al., 1981;Charlton et al., 1982; Augustine et al., 1985a,b). Several critical questions remain, however, that one would like to address with equivalent biophysical rigor at a vertebrate synapse in which the presynaptic ionic currents and postsynaptic currents can be measured simultaneously and release can be resolved at the single quantum level. In this report we take advantage of a neuromuscular synapse preparation in which this can be done.Among the important pending questions are the timing and delay between Ca 2ϩ entry during an action potential and release, the roles of different Ca and Ca-activated K (K Ca ) channels in the release process, and the quantitative relationship between Ca 2ϩ influx and release. In squid, it has been shown that Ca 2ϩ enters principally during the repolarization phase of the action potential (Llinás et al., 1982). Physiological studies of various excitable secretory cells have shown that different types of Ca channels play a dominant role in triggering release in different terminals, and often multiple Ca channel types are present in any given terminal (Kerr and Yoshikami, 1984;Pfrieger et al., 1992, Luebke et al...

Purpose Secondary lymphedema is a frequent complication of breast cancer associated with surgery, chemotherapy, or radiation following breast cancer treatment. The potential contribution of genetic susceptibility to risk of developing secondary lymphedema following surgical trauma, radiation, and other tissue insults has not been studied. Experimental Design To determine if women with breast cancer and secondary lymphedema had mutations in candidate lymphedema genes, we undertook a case - control study of 188 women diagnosed with breast cancer recruited from the University of Pittsburgh Breast Cancer Program (http://www.upmccancercenter.com/breast/index.cfm) between 2000–2010. Candidate lymphedema genes, GJC2 (encoding connexin 47 [Cx47]), FOXC2, HGF, MET, and FLT4 (encoding VEGFR3), were sequenced for mutation. Bioinformatics analysis and in vitro functional assays were used to confirm significance of novel mutations. Results Cx47 mutations were identified in individuals having secondary lymphedema following breast cancer treatment but not in breast cancer controls or normal women without breast cancer. These novel mutations are dysfunctional as assessed through in vitro assays and bioinformatics analysis, and provide evidence that altered gap junction function leads to lymphedema. Conclusions Our findings challenge the view that secondary lymphedema is solely due to mechanical trauma and support the hypothesis that genetic susceptibility is an important risk factor for secondary lymphedema. A priori recognition of genetic risk 1) raises the potential for early detection and intervention for a high risk group, and 2) allows the possibility of altering surgical approach and/or chemo- and radiation therapy, or direct medical treatment of secondary lymphedema with novel connexin modifying drugs. Translational Relevance Secondary lymphedema is a frequent and serious chronic complication of breast cancer treatment. Our finding of four independent mutations in Cx47, including one shared mutation previously reported in primary lymphedema, not only supports these mutations as a genetic risk to the development of secondary lymphedema but raises the likelihood that other genes may contribute to such a genetic risk to secondary lymphedema as well.

Neurotransmitter release is frequently regulated by peptides that modulate neuronal calcium channels. Whole-cell recordings show that the ion permeability and voltage dependence of these channels are controlled by a membrane-associated pathway involving GTP-binding proteins. Here we use perforated-patch recordings to show that, in addition to this pathway, the peptide somatostatin inhibits the calcium current in chick ciliary ganglion neurons by a second soluble pathway involving a cyclic GMP-dependent protein kinase (cGMP-PK). This somatostatin inhibition of Ca2+ current did not desensitize and was not characterized by the slowing of Ca(2+)-current activation (kinetic slowing) observed in whole-cell recordings. When cGMP-PK was inhibited, somatostatin inhibition of Ca2+ current resembled that observed with whole-cell recordings. cGMP agonists mimic the effect of somatostatin only in perforated patch recordings. An endogenous cGMP-PK therefore forms part of the mechanism by which somatostatin induces a sustained inhibition of neuronal calcium channels.