The validity of experiments based on Förster resonance energy transfer (FRET), an imaging technique widely used to measure protein-protein interactions in living cells, critically depends on the accurate and precise measurement of FRET efficiency. The use of FRET standards to determine FRET efficiency, and a consideration of such factors as how the abundance of FRET acceptors and the stoichiometry of donors and acceptors in a molecular complex can affect measured FRET efficiency, will enhance the usefulness with which FRET experiments can be interpreted.

Förster's resonance energy transfer (FRET) can be used to study protein-protein interactions in living cells. Numerous methods to measure FRET have been devised and implemented; however, the accuracy of these methods is unknown, which makes interpretation of FRET efficiency values difficult if not impossible. This problem exists due to the lack of standards with known FRET efficiencies that can be used to validate FRET measurements. The advent of spectral variants of green fluorescent protein and easy access to cell transfection technology suggests a simple solution to this problem: the development of genetic constructs with known FRET efficiencies that can be replicated with high fidelity and freely distributed. In this study, fluorescent protein constructs with progressively larger separation distances between donors and acceptors were generated and FRET efficiencies were measured using fluorescence lifetime spectroscopy, sensitized acceptor emission, and spectral imaging. Since the results from each method were in good agreement, the FRET efficiency value of each construct could be determined with high accuracy and precision, thereby justifying their use as standards.

N-type calcium channels are critical for pain transduction. Inhibitors of these channels are powerful analgesics, but clinical use of current N-type blockers remains limited by undesirable actions in other regions of the nervous system. We now demonstrate that a unique splice isoform of the N-type channel is restricted exclusively to dorsal root ganglia. By a combination of functional and molecular analyses at the single-cell level, we show that the DRG-specific exon, e37a, is preferentially present in Ca(V)2.2 mRNAs expressed in neurons that contain nociceptive markers, VR1 and Na(V)1.8. Cell-specific inclusion of exon 37a correlates closely with significantly larger N-type currents in nociceptive neurons. This unique splice isoform of the N-type channel could represent a novel target for pain management.

Protein labeling with green fluorescent protein derivatives has become an invaluable tool in cell biology. Protein quantification, however, is difficult when cells express constructs with overlapping fluorescent emissions. Under these conditions, signal separation using emission filters is inherently inefficient. Spectral imaging solves this problem by recording emission spectra directly. Unfortunately, linear unmixing, the algorithm used for quantifying individual fluorophores from emission spectra, fails when resonance energy transfer (RET) is present. We therefore sought to develop an unmixing algorithm that incorporates RET. An equation for spectral emission incorporating RET was derived and an assay based on this formalism, spectral RET (sRET), was developed. Standards with defined RET efficiencies and with known Cerulean/Venus ratios were constructed and used to test sRET. We demonstrate that sRET analysis is a comprehensive, photon-efficient method for imaging RET efficiencies and accurately determines donor and acceptor concentrations in living cells.

At its fundamental level, human memory is thought to occur at individual synaptic contact sites and manifest as persistent changes in synaptic efficacy. In digital electronics, the fundamental structure for implementing memory is the flip-flop switch, a circuit that can be triggered to flip between two stable states.

The CaV2 family of voltage-gated calcium channels, present in presynaptic nerve terminals, regulates exocytosis and synaptic transmission. Cumulative inactivation of these channels occurs during trains of action potentials, and this may control short-term dynamics at the synapse. Inactivation during brief, repetitive stimulation is primarily attributed to closed-state inactivation, and several factors modulate the susceptibility of voltage-gated calcium channels to this form of inactivation. We show that alternative splicing of an exon in a cytoplasmic region of the Ca V2.2 channel modulates its sensitivity to inactivation during trains of action potential waveforms. The presence of this exon, exon 18a, protects the Ca V 2.2 channel from entry into closed-state inactivation specifically during short (10 ms to 3 s) and small depolarizations of the membrane potential (؊60 mV to ؊50 mV). The reduced sensitivity to closed-state inactivation within this dynamic range likely underlies the differential responsiveness of Ca V2.2 splice isoforms to trains of action potential waveforms. Regulated alternative splicing of Ca V2.2 represents a possible mechanism for modulating short-term dynamics of synaptic efficacy in different regions of the nervous system. T he Ca V 2 class of voltage-gated calcium channels regulates calcium entry that triggers exocytosis from presynaptic nerve terminals (1). The temporal dynamics of calcium channel responses to action potential trains impact the fidelity of synaptic transmission (2). Membrane depolarization both activates and inactivates voltage-gated calcium channels. With repetitive, brief depolarizations, inactivation of calcium channels accumulates over time and progressively decreases calcium entry.Cumulative inactivation, a feature of both native and cloned Ca V 2 calcium channels, has been studied in detail because of its importance in regulating the short-term dynamics of synaptic efficacy (2-8). Inactivation that accumulates during brief, repetitive stimulation is thought to result from a process known as closed-state inactivation (5, 6). That is, voltage-gated calcium channels undergo inactivation in response to depolarizations that are insufficiently large to open the channel. Open-state inactivation is thought to play only a minor role in cumulative inactivation of Ca V 2 calcium channels during brief stimuli such as action potentials. Even during prolonged depolarizations that favor open-state inactivation, Ca V 2 channels inactivate with relatively slow time courses. Only in the case of Ca V 3 channels that deactivate relatively slowly following action potential repolarization has open-state inactivation been implicated in cumulative inactivation (9-11). Several factors modulate the susceptibility of voltage-gated calcium channels to cumulative inactivation, including channel subtype (6), G protein activation (3), interaction with presynaptic proteins (12, 13), and alternative splicing (10, 11). However, a limited number of studies have investigated the mechanisms by which...

Studies on the amphibian neuromuscular junction have indicated that N-type calcium channels are the sole mediators of stimulus-evoked neurotransmitter release. We show, via both presynaptic and postsynaptic voltage-clamp measurements, that dihydropyridine (DHP)-sensitive calcium channels also contribute to stimulus-evoked release at developing Xenopus neuromuscular junctions. Whereas inhibition of postsynaptic responses by omega-conotoxin (omega-Ctx) GVIA has been taken previously as evidence that only N-type channels mediate transmitter release, we find that both N-type and DHP-sensitive calcium currents are sensitive to this toxin. The unusual sensitivity of DHP-sensitive calcium channels to omega-Ctx GVIA in presynaptic terminals raises the possibility that this channel type may have escaped detection in previous physiological studies on adult frog neuromuscular junctions. Alternatively, the additional channel isoforms may be present only during early development, when they may serve to strengthen collectively presynaptic release during critical periods of synaptogenesis.

Background: Spectrally distinct fluorescent proteins (FPs) have been developed permitting the visualization of several proteins simultaneously in living cells. The emission spectra of FPs, in most cases, overlap, making signal separation based on filter technology inefficient and in cases of bleed-through, inaccurate. Spectral imaging can overcome these obstacles through a process called linear unmixing. Given a complex spectra composed of multiple fluorophores, linear unmixing can reduce the complex signal to its individual, weighted, component spectra. Spectral imaging with two-photon excitation allows the collection of nontruncated emission spectra. The accuracy of linear unmixing under these conditions needs to be evaluated. Methods: Capillaries containing defined mixtures of CFP and YFP were used to test the accuracy of linear unmixing using spectral images obtained with two-photon excitation.