Voltage-dependent Ca2+ channels play a central role in controlling neurotransmitter release at the synapse. They can be inhibited by certain G-protein-coupled receptors, acting by a pathway intrinsic to the membrane. Here we show that this inhibition results from a direct interaction between the G-protein betagamma complex and the pore-forming alpha1 subunits of several types of these channels. The interaction is mediated by the cytoplasmic linker connecting the first and second transmembrane repeats. Within this linker, binding occurs both in the alpha1 interaction domain (AID), which also mediates the interaction between the alpha1 and beta subunits of the channel, and in a second downstream sequence. Further analysis of the binding site showed that several amino-terminal residues in the AID are critical for Gbetagamma binding, defining a site distinct from the carboxy-terminal residues shown to be essential for binding the beta-subunit of the Ca2+ channel. Mutation of an arginine residue within the N-terminal motif abolished betagamma binding and rendered the channel refractory to G-protein modulation when expressed in Xenopus oocytes, showing that the interaction is indeed responsible for G-protein-dependent modulation of Ca2+ channel activity.

Recent studies on the controllability of complex systems offer a powerful mathematical framework to systematically explore the structure-function relationship in biological, social and technological networks1–3. Despite theoretical advances, we lack direct experimental proof of the validity of these widely used control principles. Here we fill this gap by applying a control framework to the connectome of the nematode C. elegans4–6, allowing us to predict the involvement of each C. elegans neuron in locomotor behaviours. We predict that control of the muscles or motor neurons requires twelve neuronal classes, which include neuronal groups previously implicated in locomotion by laser ablation7–13, as well as one previously uncharacterised neuron, PDB. We validate this prediction experimentally, finding that the ablation of PDB leads to a significant loss of dorsoventral polarity in large body bends. Importantly, control principles also allow us to investigate the involvement of individual neurons within each neuronal class. For example, we predict that, within the class of DD motor neurons, only three (DD04, DD05, or DD06) should affect locomotion when ablated individually. This prediction is also confirmed, with single-cell ablations of DD04 or DD05, but not DD02 or DD03, specifically affecting posterior body movements. Our predictions are robust to deletions of weak connections, missing connections, and rewired connections in the current connectome, indicating the potential applicability of this analytical framework to larger and less well-characterised connectomes.

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The voltage-gated calcium channel ␤ subunit is a cytoplasmic protein that stimulates activity of the channel-forming subunit, ␣ 1 , in several ways. Complementary binding sites on ␣ 1 and ␤ have been identified that are highly conserved among isoforms of the two subunits, but this interaction alone does not account for all of the functional effects of the ␤ subunit. We describe here the characterization in vitro of a second interaction, involving the carboxyl-terminal cytoplasmic domain of ␣ 1A and showing specificity for the ␤ 4 (and to a lesser extent ␤ 2a ) isoform. A deletion and chimera approach showed that the carboxyl-terminal region of ␤ 4 , poorly conserved between ␤ isoforms, contains the interaction site and plays a role in the regulation of channel inactivation kinetics. This is the first demonstration of a molecular basis for the specificity of functional effects seen for different combinations of these two channel components.Voltage-dependent calcium channels have been classified into five groups, based on their electrophysiological and pharmacological properties. L-type channels are ubiquitous, present particularly in skeletal and cardiac muscle, where they play an essential role in excitation-contraction coupling. T-type channels are important for cardiac pacemaker activity and the oscillatory activity of several thalamic neurons, while N-and P/Q-type channels are important in the control of neurotransmitter release in the central and peripheral nervous systems, and the role of R-type channels remains unclear. Two of these channels have been purified to homogeneity, the skeletal muscle L-type channel and the brain N-type channel (1, 2). Although these channels differ dramatically in function, their subunit compositions are very similar, the core subunit composition of a high voltage-activated channel consisting of an ␣ 1 subunit, the ionic pore of the channel, and two auxiliary subunits, ␤ and ␣ 2 ␦, that confer native biophysical and pharmacological properties to the channel. These subunits are encoded by at least six ␣ 1 , four ␤, and one ␣ 2 ␦ gene, for which numerous splice variants have been identified (3).The ␤ subunit is a cytoplasmic protein of 52-78 kDa that, when coexpressed with the ␣ 1 subunit, results in an increase (of up to 100-fold) in current amplitude, alteration of both the kinetics and voltage dependence of activation and inactivation, and an apparent increase in recognition sites for channelspecific toxins (e.g. see . The regulatory effects of ␤ vary in importance, depending on the combination of channel subunits studied. Although ␤ regulation seems to be highly conserved from ␤ 1 to ␤ 4 and on ␣ 1S to ␣ 1E , some important differences between these various isoforms have nevertheless been noted. The different ␤ subunits produce consistently different channel inactivation behaviors, ␤ 3 producing fast inactivation, ␤ 2 slow channel inactivation, and ␤ 1 and ␤ 4 more intermediate behaviors (9 -11). The ␤ effect also appears to be ␣ 1 isoform-dependent; the ␤-induced shift in vo...