L-type Ca2+ channels support Ca2+ entry into cells, which triggers cardiac contraction, controls hormone secretion from endocrine cells and initiates transcriptional events that support learning and memory. These channels are examples of molecular signal-transduction units that regulate themselves through their own activity. Among the many types of voltage-gated Ca2+ channel, L-type Ca2+ channels particularly display inactivation and facilitation, both of which are closely linked to the earlier entry of Ca2+ ions. Both forms of autoregulation have a significant impact on the amount of Ca2+ that enters the cell during repetitive activity, with major consequences downstream. Despite extensive biophysical analysis, the molecular basis of autoregulation remains unclear, although a putative Ca2+-binding EF-hand motif and a nearby consensus calmodulin-binding isoleucine-glutamine ('IQ') motif in the carboxy terminus of the alpha1C channel subunit have been implicated. Here we show that calmodulin is a critical Ca2+ sensor for both inactivation and facilitation, and that the nature of the modulatory effect depends on residues within the IQ motif important for calmodulin binding. Replacement of the native isoleucine by alanine removed Ca2+-dependent inactivation and unmasked a strong facilitation; conversion of the same residue to glutamate eliminated both forms of autoregulation. These results indicate that the same calmodulin molecule may act as a Ca2+ sensor for both positive and negative modulation.

concentrations, likely attained in quiescent cells. Two stretches of amino acids were found to support the tethering and to contain putative CaM-binding sequences close to or overlapping residues previously shown to affect CDI and Ca 2؉ -independent inactivation. Synthetic peptides containing these sequences displayed differences in CaM-binding properties, both in affinity and Ca 2؉ dependence, leading us to propose a novel mechanism for CDI. In contrast to a traditional disinhibitory scenario, we suggest that apoCaM is tethered at two sites and signals actively to slow inactivation. When the C-terminal lobe of CaM binds to the nearby CaM effector sequence (IQ motif), the braking effect is relieved, and CDI is accelerated.The voltage-gated L-type Ca 2ϩ channel is unique among ion channels in displaying two gating properties that are regulated by the ion that permeates the channel, calcium-dependent inactivation (CDI) 1 and calcium-dependent facilitation (CDF). These feedback mechanisms are of critical importance for regulation of the electromechanical activity of the heart and other essential physiological processes. CDI helps determine the length of the cardiac action potential plateau (1) and CDF contributes to the positive force-frequency relationship of the cardiac contraction (2).Several lines of evidence from recent work suggest that the calcium sensor mediating both of these processes may be the calcium-binding protein calmodulin (CaM). We (3) and others (4, 5) have shown that there is a Ca 2ϩ -dependent CaM-binding sequence ("IQ motif") in the cytoplasmic C-terminal tail of the pore-forming ␣ 1C subunit of the channel, within a region previously shown to confer Ca 2ϩ sensitivity (6). We have also shown that those mutations within the IQ motif that render the channel subunit unable to bind CaM also disrupt CDI (3, 7), suggesting that the IQ motif serves as the effector region for CDI. Furthermore, we (3) and others (4) have shown CDI can be blocked in a dominant negative fashion by those CaM mutants that lack Ca 2ϩ binding in their C-terminal EF-hand domains.Several important questions remain unanswered about how Ca 2ϩ and CaM might regulate L-type Ca 2ϩ channel inactivation. The first question concerns how CaM may be tethered to the L-type channel (8). There are multiple reasons for thinking that there must be a binding site that tethers the Ca 2ϩ sensor in the channel's resting state, keeping it poised for signaling as soon as Ca 2ϩ entry begins. Without tethering it would be difficult to explain the rapid development of CDI, beginning within milliseconds after L-type channel opening is initiated by depolarization (5). Tethering would also explain the dominant negative inhibitory action of mutant CaM molecules inasmuch as their binding to the tethering site would preclude binding of wild-type CaM (3, 4, 7). Recent studies have identified sequences in a cytoplasmic domain of the ␣ 1C subunit that display significant affinity for CaM even at low Ca 2ϩ concentrations (9 -11), but these do not appear to bin...

SUMMARY Specialized somatosensory neurons detect temperatures ranging from pleasantly cool or warm to burning hot and painful (nociceptive). The precise temperature ranges sensed by thermally sensitive neurons is determined by tissue specific expression of ion channels of the Transient Receptor Potential (TRP) family. We show here, that in Drosophila, TRPA1 is required for sensing of nociceptive heat. We identify two new protein isoforms of dTRPA1named dTRPA1-C and dTRPA1-D that explain this requirement. A dTRPA1-C/D reporter was exclusively expressed in nociceptors and dTRPA1-C rescued thermal nociception phenotypes when restored to mutant nociceptors. However, surprisingly, we find that dTRPA1-C is not a direct heat sensor. Alternative splicing generates at least four isoforms of dTRPA1. Our analysis of these isoforms reveals a 37 amino acid intracellular region (encoded by a single exon) that is critical for dTRPA1 temperature responses. The identification of these amino acids opens the door to a biophysical understanding of a molecular thermosensor.