Calmodulin (CaM) has been recognized as an obligate subunit for many ion channels in which its function has not been clearly established. Because channel subunits associate early during channel biosynthesis, CaM may provide a mechanism for Ca(2+)-dependent regulation of channel formation. Here we show that CaM is a constitutive component of KCNQ1 K+ channels, the most commonly mutated long-QT syndrome (LQTS) locus. CaM not only acts as a regulator of channel gating, relieving inactivation in a Ca(2+)-dependent manner, but it also contributes to control of channel assembly. Formation of functional tetramers requires CaM interaction with the KCNQ1 C-terminus. This CaM-regulated process is essential: LQTS mutants that disrupt CaM interaction prevent functional assembly of channels in a dominant-negative manner. These findings offer a new mechanism for LQTS defects and provide a basis for understanding novel ways that intracellular Ca2+ and CaM regulate ion channels.

Ca(2+)-dependent inactivation (CDI) of L-type voltage-gated Ca(2+) channels limits Ca(2+) entry into neurons, thereby regulating numerous cellular events. Here we present the isolation and purification of the Ca(2+)-sensor complex, consisting of calmodulin (CaM) and part of the channel's pore-forming alpha(1C) subunit, and demonstrate the Ca(2+)-dependent conformational shift that underlies inactivation. Dominant-negative CaM mutants that prevent CDI block the sensor's Ca(2+)-dependent conformational change. We show how Ile1654 in the CaM binding IQ motif of alpha(1C) forms the link between the Ca(2+) sensor and the downstream inactivation machinery, using the alpha(1C) EF hand motif as a signal transducer to activate the putative pore-occluder, the alpha(1C) I-II intracellular linker.

Ca2؉ has been proposed to regulate Na ؉ channels through the action of calmodulin (CaM) bound to an IQ motif or through direct binding to a paired EF hand motif in the Na v 1 C terminus. Mutations within these sites cause cardiac arrhythmias or autism, but details about how Ca 2؉ confers sensitivity are poorly understood. Studies on the homologous Ca v 1.2 channel revealed non-canonical CaM interactions, providing a framework for exploring Na ؉ channels. In contrast to previous reports, we found that Ca 2؉ does not bind directly to Na ؉ channel C termini. Rather, Ca 2؉ sensitivity appears to be mediated by CaM bound to the C termini in a manner that differs significantly from CaM regulation of Ca v 1.2. In Na v 1.2 or Na v 1.5, CaM bound to a localized region containing the IQ motif and did not support the large Ca -dependent conformational change in Na v 1.2 C terminus⅐CaM complex that was absent in the wild-type complex. In Na v 1.5, CaM modulates the Cterminal interaction with the III-IV linker, which has been suggested as necessary to stabilize the inactivation gate, to minimize sustained channel activity during depolarization, and to prevent cardiac arrhythmias that lead to sudden death. Together, these data offer new biochemical evidence for Ca 2؉

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Phototransduction in Limulus photoreceptors involves a G protein-mediated activation of phospholipase C (PLC) and subsequent steps involving InsP 3 -mediated release of intracellular Ca 2؉ . While exploring the role of calmodulin in this cascade, we found that intracellular injection of Ca 2؉ ͞calmodulin-binding peptides (CCBPs) strongly inhibited the light response. By chemically exciting the cascade at various stages, we found the primary target of this effect was not in late stages of the cascade but rather at the level of G protein and PLC. That PLC␦ 1 contains a calmodulin-like structure raised the possibility that PLC might be directly affected by CCBPs. To test this possibility, in vitro experiments were conducted on purified PLC. The activity of this enzyme was strongly inhibited by CCBPs and also inhibited by calmodulin itself. Our results suggest that the calmodulin-like region of PLC has an important role in regulating this enzyme.Phototransduction in Limulus photoreceptors is a complex excitation cascade that has sufficient amplification to produce large electrical events to single photons (1). The initial stage of this cascade resembles the rhodopsin͞G protein interaction found in vertebrate photoreceptors, but the enzyme activated by G protein is phosphoinositide-specific phospholipase C (PLC) rather than cGMP-phosphodiesterase. PLC, in turn, generates InsP 3 (2), and the resulting InsP 3 -mediated Ca 2ϩ release (3-5) leads to activation of nonspecific cation channels (2, 6), perhaps through an intermediate step involving cGMP (7,8).Invertebrate photoreceptors contain a high concentration of calmodulin (9). In the region of the photoreceptor specialized for phototransduction, the microvillar region, the concentration may be as high as 0.5 mM (10). Because the light-induced elevation of Ca 2ϩ plays an obligatory role in the excitation process in Limulus (11), we suspected that a calmodulindependent process might play an important role in a late stage of the excitation process, perhaps coupling the InsP 3 -mediated elevation of Ca 2ϩ to the opening of ion channels. If this were the case, calmodulin peptide antagonists should reduce the response to light. The experiments reported here show Ca 2ϩ ͞ calmodulin peptide antagonists do indeed have this effect. However our results show that the primary site of this effect is not at a late stage of transduction but rather an early stage involving PLC.PLC isoforms are found in all eukaryotic cells and are involved in signal transduction, including sensory, learningrelated synaptic plasticity, and oncogenesis (for reviews see refs. 12 and 13). Recently, the crystal structure of PLC␦ 1 has been obtained (14). This structure revealed EF-hand domains that resemble the structure of calmodulin with Ca 2ϩ bound (the ''calmodulin-like'' domain). Sequence alignment and ␣-helix prediction suggest the existence of similar structures in all PLC isozymes. The regulatory role of this region is unclear, but it seemed possible from our physiological results that it m...