Voltage-dependent potassium KCNQ2 (Kv7.2) channels play a prominent role in the control of neuronal excitability. These channels must associate with calmodulin to function correctly and, indeed, a mutation (R353G) that impairs this association provokes the onset of a form of human neonatal epilepsy known as benign familial neonatal convulsions (BFNC). We show here that perturbation of calmodulin binding leads to endoplasmic reticulum (ER) retention of KCNQ2, reducing the number of channels that reach the plasma membrane. Interestingly, elevating the expression of calmodulin in the BFNC mutant partially restores the intracellular distribution of the KCNQ channel. In contrast, overexpression of a Ca(2+)-binding incompetent calmodulin or sequestering of calmodulin promotes the retention of wild-type channels in the ER. Thus, a direct interaction with Ca(2+)-calmodulin appears to be critical for the correct activity of KCNQ2 potassium channels as it controls the channels' exit from the ER.

The C2 domain of protein kinase Calpha (PKCalpha) corresponds to the regulatory sequence motif, found in a large variety of membrane trafficking and signal transduction proteins, that mediates the recruitment of proteins by phospholipid membranes. In the PKCalpha isoenzyme, the Ca2+-dependent binding to membranes is highly specific to 1,2-sn-phosphatidyl-l-serine. Intrinsic Ca2+ binding tends to be of low affinity and non-cooperative, while phospholipid membranes enhance the overall affinity of Ca2+ and convert it into cooperative binding. The crystal structure of a ternary complex of the PKCalpha-C2 domain showed the binding of two calcium ions and of one 1,2-dicaproyl-sn-phosphatidyl-l-serine (DCPS) molecule that was coordinated directly to one of the calcium ions. The structures of the C2 domain of PKCalpha crystallised in the presence of Ca2+ with either 1,2-diacetyl-sn-phosphatidyl-l-serine (DAPS) or 1,2-dicaproyl-sn-phosphatidic acid (DCPA) have now been determined and refined at 1.9 A and at 2.0 A, respectively. DAPS, a phospholipid with short hydrocarbon chains, was expected to facilitate the accommodation of the phospholipid ligand inside the Ca2+-binding pocket. DCPA, with a phosphatidic acid (PA) head group, was used to investigate the preference for phospholipids with phosphatidyl-l-serine (PS) head groups. The two structures determined show the presence of an additional binding site for anionic phospholipids in the vicinity of the conserved lysine-rich cluster. Site-directed mutagenesis, on the lysine residues from this cluster that interact directly with the phospholipid, revealed a substantial decrease in C2 domain binding to vesicles when concentrations of either PS or PA were increased in the absence of Ca2+. In the complex of the C2 domain with DAPS a third Ca2+, which binds an extra phosphate group, was identified in the calcium-binding regions (CBRs). The interplay between calcium ions and phosphate groups or phospholipid molecules in the C2 domain of PKCalpha is supported by the specificity and spatial organisation of the binding sites in the domain and by the variable occupancies of ligands found in the different crystal structures. Implications for PKCalpha activity of these structural results, in particular at the level of the binding affinity of the C2 domain to membranes, are discussed.

In view of the interest shown in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) as a second messenger, we studied the activation of protein kinase C␣ by this phosphoinositide. By using two double mutants from two different sites located in the C2 domain of protein kinase C␣, we have determined and characterized the PtdIns(4,5)P 2 -binding site in the protein, which was found to be important for its activation. Thus, there are two distinct sites in the C2 domain: the first, the lysinerich cluster located in the ␤3-and ␤4-sheets and which activates the enzyme through direct binding of PtdIns(4,5)P 2 ; and the second, the already well described site formed by the Ca 2؉ -binding region, which also binds phosphatidylserine and a result of which the enzyme is activated. The results obtained in this work point to a sequential activation model, in which protein kinase C␣ needs Ca 2؉ before the PtdIns(4,5)P 2 -dependent activation of the enzyme can occur.Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) 1 plays a key role in phosphoinositide signaling and regulates a wide range of processes at many subcellular sites. It is primarily detected in the plasma membrane but is also found in secretory vesicles, lysosomes, in the endoplasmic reticulum, the Golgi, and in the nucleus (1-5). PtdIns(4,5)P 2 can either bind to intracellular proteins and directly modulate their subcellular localization and activity, or it can act as a precursor for the generation of different second messengers. For example, several families of phospholipase C enzymes are responsible for the hydrolysis of PtdIns(4,5)P 2 in cells, leading to the production of diacylglycerol and inositol 1,4,5-trisphosphate (4, 6), which may, in turn, lead to the activation of different proteins such as some PKC isotypes.Protein kinase C (PKC) composes a large family of serine/ threonine kinases, which is activated by many extracellular signals and plays a critical role in many signal-transducing pathways in the cell (7-9). Based on their enzymatic properties, the mammalian PKC isotypes have been grouped into smaller subfamilies. The first group, which includes the classical isoforms ␣, ␤I, ␤II, and ␥, can be distinguished from the other groups because its activity is regulated by diacylglycerol (DAG) and, cooperatively, by Ca 2ϩ and acidic phospholipids, particularly phosphatidylserine (PS). Members of the second group are the novel mammalian (␦, ⑀, , and ) and yeast PKCs that are not regulated by Ca 2ϩ . The third group comprises the atypical PKC isoforms, , , and , whose regulation has not been clearly established, although it is clear that they are not regulated by DAG or Ca 2ϩ (8, 10). In classical PKC isoenzymes, Ca 2ϩ -dependent binding to membranes shows a high specificity for 1,2-sn-phosphatidyl-Lserine (11)(12)(13)(14). Additionally, this group of isoenzymes is sensitive to other anionic phospholipids, including phosphatidic acid and polyphosphoinositides (15-16) and to a variety of amphipathic membrane compounds, such as arachidonic acid and fre...

The potential regulation of protein trafficking by calmodulin (CaM) is a novel concept that remains to be substantiated. We proposed that KCNQ2 K ؉ channel trafficking is regulated by CaM binding to the C-terminal A and B helices. Here we show that the L339R mutation in helix A, which is linked to human benign neonatal convulsions, perturbs CaM binding to KCNQ2 channels and prevents their correct trafficking to the plasma membrane. We used glutathione S-transferase fused to helices A and B to examine the impact of this and other mutations in helix A (I340A, I340E, A343D, and R353G) on the interaction with CaM. The process appears to require at least two steps; the first involves the transient association of CaM with KCNQ2, and in the second, the complex adopts an "active" conformation that is more stable and is that which confers the capacity to exit the endoplasmic reticulum. Significantly, the mutations that we have analyzed mainly affect the stability of the active configuration of the complex, whereas Ca 2؉ alone appears to affect the initial binding step. The spectrum of responses from this collection of mutants revealed a strong correlation between adopting the active conformation and channel trafficking in mammalian cells. These data are entirely consistent with the concept that CaM bound to KCNQ2 acts as a Ca 2؉ sensor, conferring Ca 2؉ dependence to the trafficking of the channel to the plasma membrane and fully explaining the requirement of CaM binding for KCNQ2 function.