Mechanisms of Calmodulin Regulation of Different Isoforms of Kv7.4 K+ Channels

Calmodulin (CaM), a Ca²⁺-sensing protein, is constitutively bound to IQ domains of the C termini of human Kv7 (hKv7, KCNQ) channels to mediate Ca²⁺-dependent reduction of Kv7 currents. However, the mechanism remains unclear. We report that CaM binds to two isoforms of the hKv7.4 channel in a Ca²⁺-independent manner but that only the long isoform (hKv7.4a) is regulated by Ca²⁺/CaM. Ca²⁺/CaM mediate reduction of the hKv7.4a channel by decreasing the channel open probability and altering activation kinetics. We took advantage of a known missense mutation (G321S) that has been linked to progressive hearing loss to further examine the inhibitory effects of Ca²⁺/CaM on the Kv7.4 channel. Using multidisciplinary techniques, we demonstrate that the G321S mutation may destabilize CaM binding, leading to a decrease in the inhibitory effects of Ca²⁺ on the channels. Our study utilizes an expression system to dissect the biophysical properties of the WT and mutant Kv7.4 channels. This report provides mechanistic insights into the critical roles of Ca²⁺/CaM regulation of the Kv7.4 channel under physiological and pathological conditions.     

Pivoting between Calmodulin Lobes Triggered by Calcium in the Kv7.2/Calmodulin Complex

Kv7.2 (KCNQ2) is the principal molecular component of the slow voltage gated M-channel, which strongly influences neuronal excitability. Calmodulin (CaM) binds to two intracellular C-terminal segments of Kv7.2 channels, helices A and B, and it is required for exit from the endoplasmic reticulum. However, the molecular mechanisms by which CaM controls channel trafficking are currently unknown. Here we used two complementary approaches to explore the molecular events underlying the association between CaM and Kv7.2 and their regulation by Ca²⁺. First, we performed a fluorometric assay using dansylated calmodulin (D-CaM) to characterize the interaction of its individual lobes to the Kv7.2 CaM binding site (Q2AB). Second, we explored the association of Q2AB with CaM by NMR spectroscopy, using ¹⁵N-labeled CaM as a reporter. The combined data highlight the interdependency of the N- and C-lobes of CaM in the interaction with Q2AB, suggesting that when CaM binds Ca²⁺ the binding interface pivots between the N-lobe whose interactions are dominated by helix B and the C-lobe where the predominant interaction is with helix A. In addition, Ca²⁺ makes CaM binding to Q2AB more difficult and, reciprocally, the channel weakens the association of CaM with Ca²⁺.     

Structure of a Ca2 +/CaM:Kv7.4 (KCNQ4) B-Helix Complex Provides Insight into M Current Modulation

Calmodulin (CaM) is an important regulator of Kv7.x (KCNQx) voltage-gated potassium channels. Channels from this family produce neuronal M currents and cardiac and auditory I_KS currents and harbor mutations that cause arrhythmias, epilepsy, and deafness. Despite extensive functional characterization, biochemical and structural details of the interaction between CaM and the channel have remained elusive. Here, we show that both apo-CaM and Ca^2 +/CaM bind to the C-terminal tail of the neuronal channel Kv7.4 (KCNQ4), which is involved in both hearing and mechanosensation. Interactions between apo-CaM and the Kv7.4 tail involve two C-terminal tail segments, known as the A and B segments, whereas the interaction between Ca^2 +/CaM and the Kv7.4 C-terminal tail requires only the B segment. Biochemical studies show that the calcium dependence of the CaM:B segment interaction is conserved in all Kv7 subtypes. X-ray crystallographic determination of the structure of the Ca^2 +/CaM:Kv7.4 B segment complex shows that Ca^2 +/CaM wraps around the Kv7.4 B segment, which forms an α-helix, in an antiparallel orientation that embodies a variation of the classic 1-14 Ca^2 +/CaM interaction motif. Taken together with the context of prior studies, our data suggest a model for modulation of neuronal Kv7 channels involving a calcium-dependent conformational switch from an apo-CaM form that bridges the A and B segments to a Ca^2 +/CaM form bound to the B-helix. The structure presented here also provides a context for a number of disease-causing mutations and for further dissection of the mechanisms by which CaM controls Kv7 function.     

Calmodulin binding is dispensable for Rem-mediated Ca2+ channel inhibition

GTPases of the Ras-related RGK family are negative regulators of high voltage-activated (HVA) Ca²⁺ channel activity. In this study, we examined the role of calmodulin (CaM) association in Rem-mediated Ca²⁺ channel inhibition. We found that the Rem/CaM interaction is Ca²⁺-dependent, and that truncation of the Rem C-terminus before position 277 prevents CaM binding. Serial mutagenesis of the Rem C-terminus between residues 265 and 276 to alanine generated two mutants (Rem^L271A and Rem^L274A) that displayed reduced CaM binding, and a subset of these mutants displayed significantly lower cell periphery localization than Rem^WT. However, reductions in CaM association or membrane trafficking did not affect function, as all Rem mutants could completely inhibit Ca²⁺ channels. The Rem^1–275 truncation mutant partially inhibited Ca²⁺ channel activity despite its inability to bind CaM. Taken together, these studies indicate that CaM association is not essential for either Rem-mediated Ca²⁺ channel inhibition or plasma membrane localization. Jonathan Satin is an established investigator of the American Heart Association.     

Both N- and C-lobes of calmodulin are required for Ca-dependent regulations of CaV1.2 Ca channels

We investigated the concentration- and Ca²⁺-dependent effects of CaM mutants, CaM₁₂ and CaM₃₄, in which Ca²⁺-binding to its N- and C-lobes was eliminated, respectively, on the Ca_V1.2 Ca²⁺ channel by inside-out patch clamp in guinea-pig cardiomyocytes. Both CaM₁₂ and CaM₃₄ (0.7-10 μM) applied with 3 mM ATP produced channel activity after “rundown”. Concentration-response curves were bell-shaped, similar to that for wild-type CaM. However, there was no obvious leftward shift of the curves by increasing [Ca²⁺], suggesting that both functional lobes of CaM were necessary for the Ca²⁺-dependent shift. However, channel activity induced by the CaM mutants showed Ca²⁺-dependent decrease, implying a Ca²⁺ sensor existing besides CaM. These results suggest that both N- and C-lobes of CaM are required for the Ca²⁺-dependent regulations of Ca_V1.2 Ca²⁺ channels.     

Ca2+-Calmodulin and PIP2 interactions at the proximal C-terminus of Kv7 channels

In the heart, co-assembly of Kv7.1 with KCNE1 produces the slow I_KS potassium current, which repolarizes the cardiac action potential and mutations in human Kv7.1 and KCNE1 genes cause cardiac arrhythmias. The proximal Kv7.1 C-terminus binds calmodulin (CaM) and phosphatidylinositol-4,5-bisphosphate (PIP₂) and recently we revealed the competition of PIP₂ with the calcified CaM N-lobe to a previously unidentified site in Kv7.1 helix B, also known to harbor a LQT mutation. Data indicated that PIP₂ and Ca²⁺-CaM perform the same function on I_KS channel gating to stabilize the channel open state. Here we show that similar features were observed for Kv7.1 currents expressed alone. We also find that conservation of homologous residues in helix B of other Kv7 subtypes confer similar competition of Ca²⁺-CaM with PIP2 binding to their proximal C-termini and suggest that PIP2-CaM interactions converge to Kv7 helix B to modulates channel activity in a Kv7 subtype-dependent manner.     

Two EF-hand motifs in ryanodine receptor calcium release channels contribute to isoform-specific regulation by calmodulin

The mammalian ryanodine receptor Ca²⁺ release channel (RyR) has a single conserved high affinity calmodulin (CaM) binding domain. However, the skeletal muscle RyR1 is activated and cardiac muscle RyR2 is inhibited by CaM at submicromolar Ca²⁺. This suggests isoform-specific domains are involved in RyR regulation by CaM. To gain insight into the differential regulation of cardiac and skeletal muscle RyRs by CaM, RyR1/RyR2 chimeras and mutants were expressed in HEK293 cells, and their single channel activities were measured using a lipid bilayer method. All RyR1/RyR2 chimeras and mutants were inhibited by CaM at 2 μM Ca²⁺, consistent with CaM inhibition of RyR1 and RyR2 at micromolar Ca²⁺ concentrations. An RyR1/RyR2 chimera with RyR1 N-terminal amino acid residues (aa) 1–3725 and RyR2 C-terminal aa 3692–4968 were inhibited by CaM at <1 μM Ca²⁺ similar to RyR2. In contrast, RyR1/RyR2 chimera with RyR1 aa 1–4301 and RyR2 4254–4968 was activated at <1 μM Ca²⁺ similar to RyR1. Replacement of RyR1 aa 3726–4298 with corresponding residues from RyR2 conferred CaM inhibition at <1 μM Ca²⁺, which suggests RyR1 aa 3726–4298 are required for activation by CaM. Characterization of additional RyR1/RyR2 chimeras and mutants in two predicted Ca²⁺ binding motifs in RyR1 aa 4081–4092 (EF1) and aa 4116–4127 (EF2) suggests that both EF-hand motifs and additional sequences in the large N-terminal regions are required for isoform-specific RyR1 and RyR2 regulation by CaM at submicromolar Ca²⁺ concentrations.     

EF hands at the N-lobe of calmodulin are required for both SK channel gating and stable SK–calmodulin interaction

Small conductance calcium-activated potassium (SK) channels respond to intracellular Ca²⁺ via constitutively associated calmodulin (CaM). Previous studies have proposed a modular design for the interaction between CaM and SK channels. The C-lobe and the linker of CaM are thought to regulate the constitutive binding, whereas the N-lobe binds Ca²⁺ and gates SK channels. However, we found that coexpression of mutant CaM (E/Q) where the N-lobe has only one functional EF hand leads to rapid rundown of SK channel activity, which can be recovered with exogenously applied wild-type (WT), but not mutant, CaM. Our results suggest that the mutation at the N-lobe EF hand disrupts the stable interaction between CaM and SK channel subunits, such that mutant CaM dissociates from the channel complex when the inside of the membrane is exposed to CaM-free solution. The disruption of the stable interaction does not directly result from the loss of Ca²⁺-binding capacity because SK channels and WT CaM can stably interact in the absence of Ca²⁺. These findings question a previous conclusion that CaM where the N-lobe has only one functional EF hand can stably support the gating of SK channels. They cannot be explained by the current model of modular interaction between CaM and SK channels, and they imply a role for N-lobe EF hand residues in binding to the channel subunits. Additionally, we found that a potent enhancer for SK channels, 3-oxime-6,7-dichloro-1H-indole-2,3-dione (NS309), enables the recovery of channel activity with CaM (E/Q), suggesting that NS309 stabilizes the interaction between CaM and SK channels. CaM (E/Q) can regulate Ca²⁺-dependent gating of SK channels in the presence of NS309, but with a lower apparent Ca²⁺ affinity than WT CaM.     

Calmodulin modulates insect odorant receptor function

Insect odorant receptors (ORs) are heteromeric complexes of an odor-specific receptor protein (OrX) and a ubiquitous co-receptor protein (Orco). The ORs operate as non-selective cation channels, also conducting Ca²⁺ ions. The Orco protein contains a conserved putative calmodulin (CaM)-binding motif indicating a role of CaM in its function. Using Ca²⁺ imaging to monitor OR activity we investigated the effect of CaM inhibition on the function of OR proteins. Ca²⁺ responses elicited in Drosophila olfactory sensory neurons by stimulation with the synthetic OR agonist VUAA1 were reduced and prolonged by CaM inhibition with the potent antagonist W7 but not with the weak antagonist W5. A similar effect was observed for Orco proteins heterologously expressed in CHO cells when CaM was inhibited with W7, trifluoperazine or chlorpromazine, or upon overexpression of CaM-EF-hand mutants. With the Orco CaM mutant bearing a point mutation in the putative CaM site (K339N) the Ca²⁺ responses were akin to those obtained for wild type Orco in the presence of W7. There was no uniform effect of W7 on Ca²⁺ responses in CHO cells expressing complete ORs (Or22a/Orco, Or47a/Orco, Or33a/Orco, Or56a/Orco). For Or33a and Or47a we observed no significant effect of W7, while it caused a reduced response in cells expressing Or22a and a shortened response for Or56a.     

Editorial

The inactivation of the ClC-0 chloride channel is very temperature sensitive and is greatly facilitated by the binding of a zinc ion (Zn²⁺) from the extracellular side, leading to a Zn²⁺-induced current inhibition. To further explore the relation of Zn²⁺ inhibition and the ClC-0 inactivation, we mutated all 12 cysteine amino acids in the channel and assayed the effect of Zn²⁺ on these mutants. With this approach, we found that C212 appears to be important for the sensitivity of the Zn²⁺ inhibition. Upon mutating C212 to serine or alanine, the inactivation of the channel in macroscopic current recordings disappears and the channel does not show detectable inactivation events at the single-channel level. At the same time, the channel''s sensitivity to Zn²⁺ inhibition is also greatly reduced. The other two cysteine mutants, C213G and C480S, as well as a previously identified mutant, S123T, also affect the inactivation of the channel to some degree, but the temperature-dependent inactivation process is still present, likewise the high sensitivity of the Zn²⁺ inhibition. These results further support the assertion that the inhibition of Zn²⁺ on ClC-0 is indeed due to an effect on the inactivation of the channel. The absence of inactivation in C212S mutants may provide a better defined system to study the fast gating and the ion permeation of ClC-0.     

Ethanol inhibits Kv7.2/7.3 channel open probability by reducing the PI(4,5)P2 sensitivity of Kv7.2 subunit

Ethanol often causes critical health problems by altering the neuro-nal activities of the central and peripheral nerve systems. One of the cellular targets of ethanol is the plasma membrane proteins including ion channels and receptors. Recently, we reported that ethanol elevates membrane excitability in sympathetic neurons by inhibiting Kv7.2/7.3 channels in a cell type-specific manner. Even though our studies revealed that the inhibitory effects of ethanol on the Kv7.2/7.3 channel was diminished by the increase of plasma membrane phosphatidylinositol 4,5-bisphosphate (PI (4,5)P₂), the molecular mechanism of ethanol on Kv7.2/7.3 channel inhibition remains unclear. By investigating the kinetics of Kv7.2/7.3 current in high K⁺ solution, we found that ethanol inhibited Kv7.2/7.3 channels through a mechanism distinct from that of tetraethylammonium (TEA) which enters into the pore and blocks the gate of the channels. Using a non-stationary noise analysis (NSNA), we demonstrated that the inhibitory effect of ethanol is the result of reduction of open probability (P_O) of the Kv7.2/7.3 channel, but not of a single channel current (i) or channel number (N). Finally, ethanol selectively facilitated the kinetics of Kv7.2 current suppression by voltage-sensing phosphatase (VSP)-induced PI(4,5)P₂ depletion, while it slowed down Kv7.2 current recovery from the VSP-induced inhibition. Together our results suggest that ethanol regulates neuronal activity through the reduction of open probability and PI(4,5)P₂ sensitivity of Kv7.2/7.3 channels.     

Hypoxic pulmonary vasoconstriction: role of voltage-gated potassium channels

Activity of voltage-gated potassium (Kv) channels controls membrane potential, which subsequently regulates cytoplasmic free calcium concentration ([Ca²⁺]_cyt) in pulmonary artery smooth muscle cells (PASMCs). Acute hypoxia inhibits Kv channel function in PASMCs, inducing membrane depolarization and a rise in [Ca²⁺ ]_cyt that triggers vasoconstriction. Prolonged hypoxia inhibits expression of Kv channels and reduces Kv channel currents in PASMCs. The consequent membrane depolarization raises [Ca²⁺]_cyt, thus stimulating PASMC proliferation. The present review discusses recent evidence for the involvement of Kv channels in initiation of hypoxic pulmonary vasoconstriction and in chronic hypoxia-induced pulmonary hypertension.     

Calmodulin Activation Limits the Rate of KCNQ2 K+ Channel Exit from the Endoplasmic Reticulum

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 Ca²⁺ 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 Ca²⁺ sensor, conferring Ca²⁺ dependence to the trafficking of the channel to the plasma membrane and fully explaining the requirement of CaM binding for KCNQ2 function.M-type channels are generated by the KCNQ (Kv7) family of voltage-gated subtypes (1), and they are found throughout the nervous system where they fulfill dominant roles in the control of excitability and neural discharges (2). Like all Kv channels, the KCNQ α subunits share a common core structure of six transmembrane segments with a voltage sensing domain (S1–S4) and a pore domain (S5 and S6; see Fig. 1) (3). Sequence analysis predicts the presence of four helical regions (A–D) in all family members (4), and helices A and B constitute the binding site for calmodulin (CaM).⁸ CaM is a prototypical Ca²⁺ sensor that confers Ca²⁺ sensitivity to a wide array of proteins, including ion channels (5). CaM is thought to mediate Ca²⁺-dependent inhibition of KCNQ channels (6), and in addition, we have postulated that a direct association with CaM is required for KCNQ2 channels to exit the endoplasmic reticulum (7).Open in a separate windowFIGURE 1.Topological representation of a KCNQ subunit. The consensus IQ residues are shown in bold. Circles and squares correspond to the residues mutated here (the squares indicate the mutations causing BFNC). The boxes indicate the regions with a high probability of adopting an α helix configuration, and the thick lines delineate the region fused to GST.To gain a deeper understanding into the involvement of CaM in channel trafficking, we have studied this interaction in vitro with a set of CaM-binding site-specific mutants. Although we had previously explored the impact of some of these mutants on channel trafficking (7), here we have extended this study to the mutant L339R located in helix A. This mutation, as well as the R353G mutation in helix A, has been linked to benign familial neonatal convulsions (BFNC), a human epileptic syndrome of newborn children (2). Accordingly, we demonstrate that there is a strong correlation between the impact of these mutations on the adoption of an “active” conformation by CaM and channel subunit exit from the ER, lending further support to the concept that CaM is a critical regulator for the exit of the channel from the ER.     

Activation of the Ano1 (TMEM16A) chloride channel by calcium is not mediated by calmodulin

The Ca²⁺-activated Cl channel anoctamin-1 (Ano1; Tmem16A) plays a variety of physiological roles, including epithelial fluid secretion. Ano1 is activated by increases in intracellular Ca²⁺, but there is uncertainty whether Ca²⁺ binds directly to Ano1 or whether phosphorylation or additional Ca²⁺-binding subunits like calmodulin (CaM) are required. Here we show that CaM is not necessary for activation of Ano1 by Ca²⁺ for the following reasons. (a) Exogenous CaM has no effect on Ano1 currents in inside-out excised patches. (b) Overexpression of Ca²⁺-insensitive mutants of CaM have no effect on Ano1 currents, whereas they eliminate the current mediated by the small-conductance Ca²⁺-activated K⁺ (SK2) channel. (c) Ano1 does not coimmunoprecipitate with CaM, whereas SK2 does. Furthermore, Ano1 binds very weakly to CaM in pull-down assays. (d) Ano1 is activated in excised patches by low concentrations of Ba²⁺, which does not activate CaM. In addition, we conclude that reversible phosphorylation/dephosphorylation is not required for current activation by Ca²⁺ because the current can be repeatedly activated in excised patches in the absence of ATP or other high-energy compounds. Although Ano1 is blocked by the CaM inhibitor trifluoperazine (TFP), we propose that TFP inhibits the channel in a CaM-independent manner because TFP does not inhibit Ano1 when applied to the cytoplasmic side of excised patches. These experiments lead us to conclude that CaM is not required for activation of Ano1 by Ca²⁺. Although CaM is not required for channel opening by Ca²⁺, work of other investigators suggests that CaM may have effects in modulating the biophysical properties of the channel.     

Zinc Signaling in the Hippocampus and Its Relation to Pathogenesis of Depression

Zinc is released from glutamatergic (zincergic) neuron terminals in the brain, followed by the increase in Zn²⁺ concentration in the intracellular (cytosol) compartment as well as that in the extracellular compartment. Intracellular Zn²⁺ concentration mainly increases through calcium-permeable channels and serves as Zn²⁺ signal as well as extracellular Zn²⁺ concentration. Hippocampal Zn²⁺ signaling may participate in synaptic plasticity such as long-term potentiation and cognitive function. On the other hand, subclinical zinc deficiency is common in the old who might be more susceptible to depression. Zinc deficiency causes abnormal glucocorticoid secretion and increases depression-like behavior in animals. Neuropsychological symptoms are observed prior to the decrease in Zn²⁺ signal in the hippocampus under zinc deficiency. This paper summarizes that hippocampal Zn²⁺ signaling serves to maintain healthy brain and that glucocorticoid signaling, which is responsive to zinc homeostasis in the living body, is linked to the pathophysiology of depression.     

Episodic ataxia type 1 mutation F184C alters Zn2+-induced modulation of the human K+ channel Kv1.4-Kv1.1/Kvbeta1.1

Episodic ataxia type 1 (EA1) is a Shaker-like channelopathy characterized by continuous myokymia and attacks of imbalance with jerking movements of the head, arms, and legs. Although altered expression and gating properties of Kv1.1 channels underlie EA1, several disease-causing mechanisms remain poorly understood. It is likely that Kv1.1, Kv1.4, and Kv1.1 subunits form heteromeric channels at hippocampal mossy fiber boutons from which Zn²⁺ ions are released into the synaptic cleft in a Ca²⁺-dependent fashion. The sensitivity of this macromolecular channel complex to Zn²⁺ is unknown. Here, we show that this heteromeric channel possesses a high-affinity (<10 µM) and a low-affinity (<0.5 mM) site for Zn²⁺, which are likely to regulate channel availability at distinct presynaptic membranes. Furthermore, the EA1 mutation F184C, located within the S1 segment of the Kv1.1 subunit, markedly decreased the equilibrium dissociation constants for Zn²⁺ binding to the high- and low-affinity sites. The functional characterization of the Zn²⁺ effects on heteromeric channels harboring the F184C mutation also showed that this ion significantly 1) slowed the activation rate of the channel, 2) increased the time to reach peak current amplitude, 3) decreased the rate and amount of current undergoing N-type inactivation, and 4) slowed the repriming of the channel compared with wild-type channels. These results demonstrate that the EA1 mutation F184C will not only sensitize the homomeric Kv1.1 channel to extracellular Zn²⁺, but it will also endow heteromeric channels with a higher sensitivity to this metal ion. During the vesicular release of Zn²⁺, its effects will be in addition to the intrinsic gating defects caused by the mutation, which is likely to exacerbate the symptoms by impairing the integration and transmission of signals within specific brain areas. shaker channel gating; episodic ataxia type 1; Xenopus laevis cocytes     

Regulation of ryanodine receptors by sphingosylphosphorylcholine: Involvement of both calmodulin-dependent and -independent mechanisms

Sphingosylphosphorylcholine (SPC), a lipid mediator with putative second messenger functions, has been reported to regulate ryanodine receptors (RyRs), Ca²⁺ channels of the sarco/endoplasmic reticulum. RyRs are also regulated by the ubiquitous Ca²⁺ sensor calmodulin (CaM), and we have previously shown that SPC disrupts the complex of CaM and the peptide corresponding to the CaM-binding domain of the skeletal muscle Ca²⁺ release channel (RyR1). Here we report that SPC also displaces Ca²⁺-bound CaM from the intact RyR1, which we hypothesized might lead to channel activation by relieving the negative feedback Ca²⁺CaM exerts on the channel. We could not demonstrate such channel activation as we have found that SPC has a direct, CaM-independent inhibitory effect on channel activity, confirmed by both single channel measurements and [³H]ryanodine binding assays. In the presence of Ca²⁺CaM, however, the addition of SPC did not reduce [³H]ryanodine binding, which we could explain by assuming that the direct inhibitory action of the sphingolipid was negated by the simultaneous displacement of inhibitory Ca²⁺CaM. Additional experiments revealed that RyRs are unlikely to be responsible for SPC-elicited Ca²⁺ release from brain microsomes, and that SPC does not exert detergent-like effects on sarcoplasmic reticulum vesicles. We conclude that regulation of RyRs by SPC involves both CaM-dependent and -independent mechanisms, thus, the sphingolipid might play a physiological role in RyR regulation, but channel activation previously attributed to SPC is unlikely.