Calmodulin Interaction with hEAG1 Visualized by FRET Microscopy

BACKGROUND: Ca(2+)-mediated regulation of ion channels provides a link between intracellular signaling pathways and membrane electrical activity. Intracellular Ca(2+) inhibits the voltage-gated potassium channel EAG1 through the direct binding of calmodulin (CaM). Three CaM binding sites (BD-C1: 674-683, BD-C2: 711-721, BD-N: 151-165) have been identified in a peptide screen and were proposed to mediate binding. The participation of the three sites in CaM binding to the native channel, however, remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here we studied the binding of Ca(2+)/CaM to the EAG channel by visualizing the interaction between YFP-labeled CaM and Cerulean-labeled hEAG1 in mammalian cells by FRET. The results of our cellular approach substantiate that two CaM binding sites are predominantly involved; the high-affinity 1-8-14 based CaM binding domain in the N-terminus and the second C-terminal binding domain BD-C2. Mutations at these sites completely abolished CaM binding to hEAG1. CONCLUSIONS/SIGNIFICANCE: We demonstrated that the BD-N and BD-C2 binding domains are sufficient for CaM binding to the native channel, and, therefore, that BD-C1 is unable to bind CaM independently

Selective Interaction of Syntaxin 1A with KCNQ2: Possible Implications for Specific Modulation of Presynaptic Activity

KCNQ2/KCNQ3 channels are the molecular correlates of the neuronal M-channels, which play a major role in the control of neuronal excitability. Notably, they differ from homomeric KCNQ2 channels in their distribution pattern within neurons, with unique expression of KCNQ2 in axons and nerve terminals. Here, combined reciprocal coimmunoprecipitation and two-electrode voltage clamp analyses in Xenopus oocytes revealed a strong association of syntaxin 1A, a major component of the exocytotic SNARE complex, with KCNQ2 homomeric channels resulting in a ∼2-fold reduction in macroscopic conductance and ∼2-fold slower activation kinetics. Remarkably, the interaction of KCNQ2/Q3 heteromeric channels with syntaxin 1A was significantly weaker and KCNQ3 homomeric channels were practically resistant to syntaxin 1A. Analysis of different KCNQ2 and KCNQ3 chimeras and deletion mutants combined with in-vitro binding analysis pinpointed a crucial C-terminal syntaxin 1A-association domain in KCNQ2. Pull-down and coimmunoprecipitation analyses in hippocampal and cortical synaptosomes demonstrated a physical interaction of brain KCNQ2 with syntaxin 1A, and confocal immunofluorescence microscopy showed high colocalization of KCNQ2 and syntaxin 1A at presynaptic varicosities. The selective interaction of syntaxin 1A with KCNQ2, combined with a numerical simulation of syntaxin 1A's impact in a firing-neuron model, suggest that syntaxin 1A's interaction is targeted at regulating KCNQ2 channels to fine-tune presynaptic transmitter release, without interfering with the function of KCNQ2/3 channels in neuronal firing frequency adaptation

Inositol-1,4,5-trisphosphate receptor-mediated Ca2+ waves in pyramidal neuron dendrites propagate through hot spots and cold spots

We studied inositol-1,4,5-trisphosphate (IP3) receptor-dependent intracellular Ca2+ waves in CA1 hippocampal and layer V medial prefrontal cortical pyramidal neurons using whole-cell patch-clamp recordings and Ca2+ fluorescence imaging. We observed that Ca2+ waves propagate in a saltatory manner through dendritic regions where increases in the intracellular concentration of Ca2+ ([Ca2+]i) were large and fast (‘hot spots’) separated by regions where increases in [Ca2+]i were comparatively small and slow (‘cold spots’). We also observed that Ca2+ waves typically initiate in hot spots and terminate in cold spots, and that most hot spots, but few cold spots, are located at dendritic branch points. Using immunohistochemistry, we found that IP3 receptors (IP3Rs) are distributed in clusters along pyramidal neuron dendrites and that the distribution of inter-cluster distances is nearly identical to the distribution of inter-hot spot distances. These findings support the hypothesis that the dendritic locations of Ca2+ wave hot spots in general, and branch points in particular, are specially equipped for regenerative IP3R-dependent internal Ca2+ release. Functionally, the observation that IP3R-dependent [Ca2+]i rises are greater at branch points raises the possibility that this novel Ca2+ signal may be important for the regulation of Ca2+-dependent processes in these locations. Futhermore, the observation that Ca2+ waves tend to fail between hot spots raises the possibility that influences on Ca2+ wave propagation may determine the degree of functional association between distinct Ca2+-sensitive dendritic domains