Phospholemman Modulates the Gating of Cardiac L-Type Calcium Channels

Ca²⁺ entry through L-type calcium channels (Ca_V1.2) is critical in shaping the cardiac action potential and initiating cardiac contraction. Modulation of Ca_V1.2 channel gating directly affects myocyte excitability and cardiac function. We have found that phospholemman (PLM), a member of the FXYD family and regulator of cardiac ion transport, coimmunoprecipitates with Ca_V1.2 channels from guinea pig myocytes, which suggests PLM is an endogenous modulator. Cotransfection of PLM in HEK293 cells slowed Ca_V1.2 current activation at voltages near the threshold for activation, slowed deactivation after long and strong depolarizing steps, enhanced the rate and magnitude of voltage-dependent inactivation (VDI), and slowed recovery from inactivation. However, Ca²⁺-dependent inactivation was not affected. Consistent with slower channel closing, PLM significantly increased Ca²⁺ influx via Ca_V1.2 channels during the repolarization phase of a human cardiac action potential waveform. Our results support PLM as an endogenous regulator of Ca_V1.2 channel gating. The enhanced VDI induced by PLM may help protect the heart under conditions such as ischemia or tachycardia where the channels are depolarized for prolonged periods of time and could induce Ca²⁺ overload. The time and voltage-dependent slowed deactivation could represent a gating shift that helps maintain Ca²⁺ influx during the cardiac action potential waveform plateau phase.     

The HOOK region of β subunits controls gating of voltage-gated Ca2+ channels by electrostatically interacting with plasma membrane

Recently, we showed that the HOOK region of the β2 subunit electrostatically interacts with the plasma membrane and regulates the current inactivation and phosphatidylinositol 4,5-bisphosphate (PIP₂) sensitivity of voltage-gated Ca²⁺ (Ca_V) 2.2 channels. Here, we report that voltage-dependent gating and current density of the Ca_V2.2 channels are also regulated by the HOOK region of the β2 subunit. The HOOK region can be divided into 3 domains: S (polyserine), A (polyacidic), and B (polybasic). We found that the A domain shifted the voltage-dependent inactivation and activation of Ca_V2.2 channels to more hyperpolarized and depolarized voltages, respectively, whereas the B domain evoked these responses in the opposite directions. In addition, the A domain decreased the current density of the Ca_V2.2 channels, while the B domain increased it. Together, our data demonstrate that the flexible HOOK region of the β2 subunit plays an important role in determining the overall Ca_V channel gating properties.     

Structural and biophysical determinants of single CaV3.1 and CaV3.2 T-type calcium channel inhibition by N2O

We investigated the biophysical mechanism of inhibition of recombinant T-type calcium channels Ca_V3.1 and Ca_V3.2 by nitrous oxide (N₂O). To identify functionally important channel structures, chimeras with reciprocal exchange of the N-terminal domains I and II and C-terminal domains III and IV were examined. In whole-cell recordings N₂O significantly inhibited Ca_V3.2, and – less pronounced – Ca_V3.1. A Ca_V3.2-prevalent inhibition of peak currents was also detected in cell-attached multi-channel patches. In cell-attached patches containing ≤3 channels N₂O reduced average peak current of Ca_V3.2 by decreasing open probability and open time duration. Effects on Ca_V3.1 were smaller and mediated by a reduced fraction of sweeps containing channel activity. Without drug, single Ca_V3.1 channels were significantly less active than Ca_V3.2. Chimeras revealed that domains III and IV control basal gating properties. Domains I and II, in particular a histidine residue within Ca_V3.2 (H191), are responsible for the subtype-prevalent N₂O inhibition. Our study demonstrates the biophysical (open times, open probability) and structural (domains I and II) basis of action of N₂O on Ca_V3.2. Such a fingerprint of single channels can help identifying the molecular nature of native channels. This is exemplified by a characterization of single channels expressed in human hMTC cells as functional homologues of recombinant Ca_V3.1.     

Multiscale modeling shows that dielectric differences make NaV channels faster than KV channels

The generation of action potentials in excitable cells requires different activation kinetics of voltage-gated Na (Na_V) and K (K_V) channels. Na_V channels activate much faster and allow the initial Na⁺ influx that generates the depolarizing phase and propagates the signal. Recent experimental results suggest that the molecular basis for this kinetic difference is an amino acid side chain located in the gating pore of the voltage sensor domain, which is a highly conserved isoleucine in K_V channels but an equally highly conserved threonine in Na_V channels. Mutagenesis suggests that the hydrophobicity of this side chain in Shaker K_V channels regulates the energetic barrier that gating charges cross as they move through the gating pore and control the rate of channel opening. We use a multiscale modeling approach to test this hypothesis. We use high-resolution molecular dynamics to study the effect of the mutation on polarization charge within the gating pore. We then incorporate these results in a lower-resolution model of voltage gating to predict the effect of the mutation on the movement of gating charges. The predictions of our hierarchical model are fully consistent with the tested hypothesis, thus suggesting that the faster activation kinetics of Na_V channels comes from a stronger dielectric polarization by threonine (Na_V channel) produced as the first gating charge enters the gating pore compared with isoleucine (K_V channel).     

Impact of gating modulation in CaV1.3 L-type calcium channels

Ca_V1.3 L-type channels control inner hair cell (IHC) sensory and sinoatrial node (SAN) function, and excitability in central neurons by means of their low-voltage activation and inactivation properties. In SAN cells Ca_V1.3 inward calcium current (I_Ca) inactivates rapidly whereas in IHCs inactivation is slow. A candidate suggested in slowing Ca_V1.3 channel inactivation is the presynaptically located ribbon-synapse protein RIM that is expressed in immature IHCs in presynaptic compartments also expressing Ca_V1.3 channels. Ca_V1.3 channel gating is also modulated by an intramolecular C-terminal mechanism. This mechanism was elicited during analysis of human C-terminal splice variants that differ in the length of their C-terminus and that modulates the channel’s negative activation range and slows calcium-dependent inactivation.     

Excitation-Contraction Coupling: The distinct role of the four voltage sensors of the skeletal CaV1.1 channel in voltage-dependent activation

Initiation of skeletal muscle contraction is triggered by rapid activation of RYR1 channels in response to sarcolemmal depolarization. RYR1 is intracellular and has no voltage-sensing structures, but it is coupled with the voltage-sensing apparatus of Ca_V1.1 channels to inherit voltage sensitivity. Using an opto-electrophysiological approach, we resolved the excitation-driven molecular events controlling both Ca_V1.1 and RYR1 activations, reported as fluorescence changes. We discovered that each of the four human Ca_V1.1 voltage-sensing domains (VSDs) exhibits unique biophysical properties: VSD-I time-dependent properties were similar to ionic current activation kinetics, suggesting a critical role of this voltage sensor in Ca_V1.1 activation; VSD-II, VSD-III, and VSD-IV displayed faster activation, compatible with kinetics of sarcoplasmic reticulum Ca²⁺ release. The prominent role of VSD-I in governing Ca_V1.1 activation was also confirmed using a naturally occurring, charge-neutralizing mutation in VSD-I (R174W). This mutation abolished Ca_V1.1 current at physiological membrane potentials by impairing VSD-I activation without affecting the other VSDs. Using a structurally relevant allosteric model of Ca_V activation, which accounted for both time- and voltage-dependent properties of Ca_V1.1, to predict VSD-pore coupling energies, we found that VSD-I contributed the most energy (~75 meV or ∼3 kT) toward the stabilization of the open states of the channel, with smaller (VSD-IV) or negligible (VSDs II and III) energetic contribution from the other voltage sensors (<25 meV or ∼1 kT). This study settles the longstanding question of how Ca_V1.1, a slowly activating channel, can trigger RYR1 rapid activation, and reveals a new mechanism for voltage-dependent activation in ion channels, whereby pore opening of human Ca_V1.1 channels is primarily driven by the activation of one voltage sensor, a mechanism distinct from that of all other voltage-gated channels.     

Functional properties of the Cav1.2 calcium channel activated by calmodulin in the absence of α2δ subunits

Voltage-activated Ca_v1.2 calcium channels require association of the pore-forming α_1C subunit with accessory Ca_vβ and α₂δ subunits. Binding of a single calmodulin (CaM) to α_1C supports Ca²⁺-dependent inactivation (CDI). The human Ca_v1.2 channel is silent in the absence of Ca_vβ and/or α₂δ. Recently, we found that coexpression of exogenous CaM (CaM_ex) supports plasma membrane targeting, gating facilitation and CDI of the channel in the absence of Ca_vβ. Here we discovered that CaM_ex and its Ca²⁺-insensitive mutant (CaM₁₂₃₄) rendered active α_1C/Ca_vβ channel in the absence of α₂δ. Coexpression of CaM_ex with α_1C and β_2d in calcium-channel-free COS-1 cells recovered gating of the channel and supported CDI. Voltage-dependence of activation was shifted by ≈ +40 mV to depolarization potentials. The calcium current reached maximum at +40 mV (20 mM Ca²⁺) and exhibited approximately 3 times slower activation and 5 times slower inactivation kinetics compared to the wild-type channel. Furthermore, both CaM_ex and CaM₁₂₃₄ accelerated recovery from inactivation and induced facilitation of the calcium current by strong depolarization prepulse, the properties absent from the human vascular/neuronal Ca_v1.2 channel. The data suggest a previously unknown action of CaM that in the presence of Ca_vβ translates into activation of the α₂δ-deficient calcium channel and alteration of its properties.     

Cooperative Activation of the T-type CaV3.2 Channel: INTERACTION BETWEEN DOMAINS II AND III*

T-type Ca_V3 channels are important mediators of Ca²⁺ entry near the resting membrane potential. Little is known about the molecular mechanisms responsible for channel activation. Homology models based upon the high-resolution structure of bacterial Na_V channels predict interaction between the S4-S5 helix of Domain II (IIS4-S5) and the distal S6 pore region of Domain II (IIS6) and Domain III (IIIS6). Functional intra- and inter-domain interactions were investigated with a double mutant cycle analysis. Activation gating and channel kinetics were measured for 47 single mutants and 20 pairs of mutants. Significant coupling energies (ΔΔG_interact ≥ 1.5 kcal mol⁻¹) were measured for 4 specific pairs of mutants introduced between IIS4-S5 and IIS6 and between IIS4-S5 and IIIS6. In agreement with the computer based models, Thr-911 in IIS4-S5 was functionally coupled with Ile-1013 in IIS6 during channel activation. The interaction energy was, however, found to be stronger between Val-907 in IIS4-S5 and Ile-1013 in IIS6. In addition Val-907 was significantly coupled with Asn-1548 in IIIS6 but not with Asn-1853 in IVS6. Altogether, our results demonstrate that the S4-S5 and S6 helices from adjacent domains are energetically coupled during the activation of a low voltage-gated T-type Ca_V3 channel.     

Inactivation gating of Kv7.1 channels does not involve concerted cooperative subunit interactions

Inactivation is an intrinsic property of numerous voltage-gated K⁺ (Kv) channels and can occur by N-type or/and C-type mechanisms. N-type inactivation is a fast, voltage independent process, coupled to activation, with each inactivation particle of a tetrameric channel acting independently. In N-type inactivation, a single inactivation particle is necessary and sufficient to occlude the pore. C-type inactivation is a slower process, involving the outermost region of the pore and is mediated by a concerted, highly cooperative interaction between all four subunits. Inactivation of Kv7.1 channels does not exhibit the hallmarks of N- and C-type inactivation. Inactivation of WT Kv7.1 channels can be revealed by hooked tail currents that reflects the recovery from a fast and voltage-independent inactivation process. However, several Kv7.1 mutants such as the pore mutant L273F generate an additional voltage-dependent slow inactivation. The subunit interactions during this slow inactivation gating remain unexplored. The goal of the present study was to study the nature of subunit interactions along Kv7.1 inactivation gating, using concatenated tetrameric Kv7.1 channel and introducing sequentially into each of the four subunits the slow inactivating pore mutation L273F. Incorporating an incremental number of inactivating mutant subunits did not affect the inactivation kinetics but slowed down the recovery kinetics from inactivation. Results indicate that Kv7.1 inactivation gating is not compatible with a concerted cooperative process. Instead, adding an inactivating subunit L273F into the Kv7.1 tetramer incrementally stabilizes the inactivated state, which suggests that like for activation gating, Kv7.1 slow inactivation gating is not a concerted process.     

Contrasting Effects of Cd<Superscript>2+</Superscript> and Co<Superscript>2+</Superscript> on the Blocking/Unblocking of Human Ca<Subscript>v</Subscript>3 Channels

Inorganic ions have been used widely to investigate biophysical properties of high voltage-activated calcium channels (HVA: Ca_v1 and Ca_v2 families). In contrast, such information regarding low voltage-activated calcium channels (LVA: Ca_v3 family) is less documented. We have studied the blocking effect of Cd²⁺, Co²⁺ and Ni²⁺ on T-currents expressed by human Ca_v3 channels: Ca_v3.1, Ca_v3.2, and Ca_v3.3. With the use of the whole-cell configuration of the patch-clamp technique, we have recorded Ca²⁺ (2 mM) currents from HEK−293 cells stably expressing recombinant T-type channels. Cd²⁺ and Co²⁺ block was 2- to 3-fold more potent for Ca_v3.2 channels (EC₅₀ = 65 and 122 μM, respectively) than for the other two LVA channel family members. Current-voltage relationships indicate that Co²⁺ and Ni²⁺ shift the voltage dependence of Ca_v3.1 and Ca_v3.3 channels activation to more positive potentials. Interestingly, block of those two Ca_v3 channels by Co²⁺ and Ni²⁺ was drastically increased at extreme negative voltages; in contrast, block due to Cd²⁺ was significantly decreased. This unblocking effect was slightly voltage-dependent. Tail-current analysis reveals a differential effect of Cd²⁺ on Ca_v3.3 channels, which can not close while the pore is occupied with this metal cation. The results suggest that metal cations affect differentially T-type channel activity by a mechanism involving the ionic radii of inorganic ions and structural characteristics of the channels pore.     

Functional Expression of T-Type Ca2+ Channels in Spinal Motoneurons of the Adult Turtle

Voltage-gated Ca²⁺ (Ca_V) channels are transmembrane proteins comprising three subfamilies named Ca_V1, Ca_V2 and Ca_V3. The Ca_V3 channel subfamily groups the low-voltage activated Ca²⁺ channels (LVA or T-type) a significant role in regulating neuronal excitability. Ca_V3 channel activity may lead to the generation of complex patterns of action potential firing such as the postinhibitory rebound (PIR). In the adult spinal cord, these channels have been found in dorsal horn interneurons where they control physiological events near the resting potential and participate in determining excitability. In motoneurons, Ca_V3 channels have been found during development, but their functional expression has not yet been reported in adult animals. Here, we show evidence for the presence of Ca_V3 channel-mediated PIR in motoneurons of the adult turtle spinal cord. Our results indicate that Ni²⁺ and NNC55-0396, two antagonists of Ca_V3 channel activity, inhibited PIR in the adult turtle spinal cord. Molecular biology and biochemical assays revealed the expression of the Ca_V3.1 channel isotype and its localization in motoneurons. Together, these results provide evidence for the expression of Ca_V3.1 channels in the spinal cord of adult animals and show also that these channels may contribute to determine the excitability of motoneurons.     

Mechanisms of a Human Skeletal Myotonia Produced by Mutation in the C-Terminus of NaV1.4: Is Ca2+ Regulation Defective?

Mutations in the cytoplasmic tail (CT) of voltage gated sodium channels cause a spectrum of inherited diseases of cellular excitability, yet to date only one mutation in the CT of the human skeletal muscle voltage gated sodium channel (hNa_V1.4_F1705I) has been linked to cold aggravated myotonia. The functional effects of altered regulation of hNa_V1.4_F1705I are incompletely understood. The location of the hNa_V1.4_F1705I in the CT prompted us to examine the role of Ca²⁺ and calmodulin (CaM) regulation in the manifestations of myotonia. To study Na channel related mechanisms of myotonia we exploited the differences in rat and human Na_V1.4 channel regulation by Ca²⁺ and CaM. hNa_V1.4_F1705I inactivation gating is Ca²⁺-sensitive compared to wild type hNa_V1.4 which is Ca²⁺ insensitive and the mutant channel exhibits a depolarizing shift of the V_1/2 of inactivation with CaM over expression. In contrast the same mutation in the rNa_V1.4 channel background (rNa_V1.4_F1698I) eliminates Ca²⁺ sensitivity of gating without affecting the CaM over expression induced hyperpolarizing shift in steady-state inactivation. The differences in the Ca²⁺ sensitivity of gating between wild type and mutant human and rat Na_V1.4 channels are in part mediated by a divergence in the amino acid sequence in the EF hand like (EFL) region of the CT. Thus the composition of the EFL region contributes to the species differences in Ca²⁺/CaM regulation of the mutant channels that produce myotonia. The myotonia mutation F1705I slows I_Na decay in a Ca²⁺-sensitive fashion. The combination of the altered voltage dependence and kinetics of I_Na decay contribute to the myotonic phenotype and may involve the Ca²⁺-sensing apparatus in the CT of Na_V1.4.     

Single mutations strongly alter the K-selective pore of the Kin channel KAT1

Voltage-dependent potassium uptake channels represent the major pathway for K⁺ accumulation underlying guard cell swelling and stomatal opening. The core structure of these Shaker-like channels is represented by six transmembrane domains and an amphiphilic pore-forming region between the fifth and sixth domain. To explore the effect of point mutations within the stretch of amino acids lining the K⁺ conducting pore of KAT1, an Arabidopsis thaliana guard cell K_in channel, we selected residues deep inside and in the periphery of the pore. The mutations on positions 256 and 267 strongly altered the interaction of the permeation pathway with external Ca²⁺ ions. Point mutations on position 256 in KAT1 affected the affinity towards Ca²⁺, the voltage dependence as well as kinetics of the Ca²⁺ blocking reaction. Among these T256S showed a Ca²⁺ phenotype reminiscent of an inactivation-like process, a phenomenon unknown for K_in channels so far. Mutating histidine 267 to alanine, a substitution strongly affecting C-type inactivation in Shaker, this apparent inactivation could be linked to a very slow calcium block. The mutation H267A did not affect gating but hastened the Ca²⁺ block/unblock kinetics and increased the Ca²⁺ affinity of KAT1. From the analysis of the presented data we conclude that even moderate point mutations in the pore of KAT1 seem to affect the pore geometry rather than channel gating.     

Distinct roles for CaV1.1’s voltage-sensing domains

Study reveals how a slowly activating calcium channel is able to control rapid excitation–contraction coupling in skeletal muscle.

Skeletal muscle contraction is initiated by action potentials that depolarize the muscle fiber and trigger the rapid release of Ca²⁺ from the SR via RYR1 channels. This process of excitation–contraction coupling depends on voltage-gated Ca_V1.1 channels in the plasma membrane, or sarcolemma, of muscle fibers. But Ca_V1.1 channels are only slowly activated by changes in the sarcolemma membrane potential, and it is therefore unclear how they are able to trigger the much faster activation of RYR1 channels. In this issue of JGP, Savalli et al. reveal that this paradox can be explained by the fact that each of Ca_V1.1’s four voltage-sensing domains (VSDs) have distinct biophysical properties (1).Nicoletta Savalli (left), Riccardo Olcese (center), and colleagues reveal the distinct physical properties of the Ca_V1.1 channel’s four voltage-sensing domains (VSD I–IV, right). VSD-I shows slow activation kinetics and is the main contributor to the opening of Ca_V1.1. The other VSDs activate much faster and may therefore be coupled to RYR1 to mediate the rapid release of Ca²⁺ from the SR during skeletal muscle contraction.RYR1 channels have no voltage-sensing machinery of their own and therefore rely on a physical connection to Ca_V1.1 channels to release Ca²⁺ and initiate muscle contraction in response to muscle fiber depolarization. But RYR1 channels open ∼25 times faster than Ca_V1.1 channels. “So, how can these slowly activating Ca_V1.1 channels trigger the rapid release of Ca²⁺ from the SR?” asks Riccardo Olcese, a professor at the David Geffen School of Medicine, UCLA.Olcese and colleagues, including Assistant Project Scientist Nicoletta Savalli, suspected that the answer might lie in the fact that, like many other voltage-gated ion channels, Ca_V1.1 has four VSDs that alter their conformation in response to voltage changes. These domains are similar, but not identical, to each other, potentially enabling them to have distinct biophysical properties and perform distinct functions. Indeed, Olcese and colleagues previously demonstrated that, in the closely related channel Ca_V1.2, only VSDs II and III are involved in pore opening (2, 3).Savalli et al. used voltage-clamp fluorometry to compare the properties of Ca_V1.1’s VSDs, expressing the channel in Xenopus oocytes and labeling each of its VSDs in turn with an environmentally sensitive fluorophore to report voltage-dependent changes in their conformation (1). “We found that the four VSDs were very heterogenous in both their kinetics and voltage dependencies,” says Olcese. “VSD-I had very slow kinetics, compatible with the slow activation of the Ca_V1.1 pore. The other three VSDs had much faster kinetics and could, therefore, be good candidates to be the voltage sensors for RYR1 activation.”Olcese and colleagues confirmed the importance of VSD-I for Ca_V1.1 activation by analyzing a naturally occurring, charge-neutralizing mutation in this domain, R174W, that is linked to malignant hyperthermia (4). The team found that this mutation reduced the voltage-sensitivity of VSD-I and abolished the ability of Ca_V1.1 to conduct Ca²⁺ at physiological membrane potentials, but had no effect on the behavior of the other three VSDs.Finally, Savalli et al. applied their data on both the wild-type and mutant VSDs to an allosteric model of Ca_V activation (2, 3), which predicted that VSD-I contributes most of the energy required to stabilize the open state of Ca_V1.1, while the other VSDs contribute little to nothing.Thus, Ca_V1.1 activation is mainly driven by a single VSD—a mechanism that hasn’t been seen in any other voltage-gated ion channel—leaving the other VSDs free to perform other functions, such as the rapid activation of RYR1. Olcese and colleagues now want to pinpoint exactly which VSD(s) are coupled to RYR1 and determine how they trigger rapid Ca²⁺ release from the SR.     

Conserved biophysical features of the CaV2 presynaptic Ca2+ channel homologue from the early-diverging animal Trichoplax adhaerens

The dominant role of Ca_V2 voltage-gated calcium channels for driving neurotransmitter release is broadly conserved. Given the overlapping functional properties of Ca_V2 and Ca_V1 channels, and less so Ca_V3 channels, it is unclear why there have not been major shifts toward dependence on other Ca_V channels for synaptic transmission. Here, we provide a structural and functional profile of the Ca_V2 channel cloned from the early-diverging animal Trichoplax adhaerens, which lacks a nervous system but possesses single gene homologues for Ca_V1–Ca_V3 channels. Remarkably, the highly divergent channel possesses similar features as human Ca_V2.1 and other Ca_V2 channels, including high voltage–activated currents that are larger in external Ba²⁺ than in Ca²⁺; voltage-dependent kinetics of activation, inactivation, and deactivation; and bimodal recovery from inactivation. Altogether, the functional profile of Trichoplax Ca_V2 suggests that the core features of presynaptic Ca_V2 channels were established early during animal evolution, after Ca_V1 and Ca_V2 channels emerged via proposed gene duplication from an ancestral Ca_V1/2 type channel. The Trichoplax channel was relatively insensitive to mammalian Ca_V2 channel blockers ω-agatoxin-IVA and ω-conotoxin-GVIA and to metal cation blockers Cd²⁺ and Ni²⁺. Also absent was the capacity for voltage-dependent G-protein inhibition by co-expressed Trichoplax Gβγ subunits, which nevertheless inhibited the human Ca_V2.1 channel, suggesting that this modulatory capacity evolved via changes in channel sequence/structure, and not G proteins. Last, the Trichoplax channel was immunolocalized in cells that express an endomorphin-like peptide implicated in cell signaling and locomotive behavior and other likely secretory cells, suggesting contributions to regulated exocytosis.     

Single-Channel Resolution of the Interaction between C-Terminal CaV1.3 Isoforms and Calmodulin

Voltage-dependent calcium (Ca_V) 1.3 channels are involved in the control of cellular excitability and pacemaking in neuronal, cardiac, and sensory cells. Various proteins interact with the alternatively spliced channel C-terminus regulating gating of Ca_V1.3 channels. Binding of a regulatory calcium-binding protein calmodulin (CaM) to the proximal C-terminus leads to the boosting of channel activity and promotes calcium-dependent inactivation (CDI). The C-terminal modulator domain (CTM) of Ca_V1.3 channels can interfere with the CaM binding, thereby inhibiting channel activity and CDI. Here, we compared single-channel gating behavior of two natural Ca_V1.3 splice isoforms: the long Ca_V1.3₄₂ with the full-length CTM and the short Ca_V1.3_42A with the C-terminus truncated before the CTM. We found that CaM regulation of Ca_V1.3 channels is dynamic on a minute timescale. We observed that at equilibrium, single Ca_V1.3₄₂ channels occasionally switched from low to high open probability, which perhaps reflects occasional binding of CaM despite the presence of CTM. Similarly, when the amount of the available CaM in the cell was reduced, the short Ca_V1.3_42A isoform showed patterns of the low channel activity. CDI also underwent periodic changes with corresponding kinetics in both isoforms. Our results suggest that the competition between CTM and CaM is influenced by calcium, allowing further fine-tuning of Ca_V1.3 channel activity for particular cellular needs.     

Divergent Ca2+/calmodulin feedback regulation of CaV1 and CaV2 voltage-gated calcium channels evolved in the common ancestor of Placozoa and Bilateria

Ca_V1 and Ca_V2 voltage-gated calcium channels evolved from an ancestral Ca_V1/2 channel via gene duplication somewhere near the stem animal lineage. The divergence of these channel types led to distinguishing functional properties that are conserved among vertebrates and bilaterian invertebrates and contribute to their unique cellular roles. One key difference pertains to their regulation by calmodulin (CaM), wherein bilaterian Ca_V1 channels are uniquely subject to pronounced, buffer-resistant Ca²⁺/CaM-dependent inactivation, permitting negative feedback regulation of calcium influx in response to local cytoplasmic Ca²⁺ rises. Early diverging, nonbilaterian invertebrates also possess Ca_V1 and Ca_V2 channels, but it is unclear whether they share these conserved functional features. The most divergent animals to possess both Ca_V1 and Ca_V2 channels are placozoans such as Trichoplax adhaerens, which separated from other animals over 600 million years ago shortly after their emergence. Hence, placozoans can provide important insights into the early evolution of Ca_V1 and Ca_V2 channels. Here, we build upon previous characterization of Trichoplax Ca_V channels by determining the cellular expression and ion-conducting properties of the Ca_V1 channel orthologue, TCa_V1. We show that TCa_V1 is expressed in neuroendocrine-like gland cells and contractile dorsal epithelial cells. In vitro, this channel conducts dihydropyridine-insensitive, high-voltage–activated Ca²⁺ currents with kinetics resembling those of rat Ca_V1.2 but with left-shifted voltage sensitivity for activation and inactivation. Interestingly, TCa_V1, but not TCa_V2, exhibits buffer-resistant Ca²⁺/CaM-dependent inactivation, indicating that this functional divergence evolved prior to the emergence of bilaterian animals and may have contributed to their unique adaptation for cytoplasmic Ca²⁺ signaling within various cellular contexts.     

Phosphorylation states greatly regulate the activity and gating properties of Cav3.1 T-type Ca2+ channels

Ca_v3.1 T-type Ca²⁺ channels play pivotal roles in neuronal low-threshold spikes, visceral pain, and pacemaker activity. Phosphorylation has been reported to potently regulate the activity and gating properties of Ca_v3.1 channels. However, systematic identification of phosphorylation sites (phosphosites) in Ca_v3.1 channel has been poorly investigated. In this work, we analyzed rat Ca_v3.1 protein expressed in HEK-293 cells by mass spectrometry, identified 30 phosphosites located at the cytoplasmic regions, and illustrated them as a Ca_v3.1 phosphorylation map which includes the reported mouse Ca_v3.1 phosphosites. Site-directed mutagenesis of the phosphosites to Ala residues and functional analysis of the phospho-silent Ca_v3.1 mutants expressed in Xenopus oocytes showed that the phospho-silent mutation of the N-terminal Ser18 reduced its current amplitude with accelerated current kinetics and negatively shifted channel availability. Remarkably, the phospho-silent mutations of the C-terminal Ser residues (Ser1924, Ser2001, Ser2163, Ser2166, or Ser2189) greatly reduced their current amplitude without altering the voltage-dependent gating properties. In contrast, the phosphomimetic Asp mutations of Ca_v3.1 on the N- and C-terminal Ser residues reversed the effects of the phospho-silent mutations. Collectively, these findings demonstrate that the multiple phosphosites of Ca_v3.1 at the N- and C-terminal regions play crucial roles in the regulation of the channel activity and voltage-dependent gating properties.     

Of cilia,titin, and neurosteroids

Missense mutations at arginine residues in the S4 voltage-sensor domains of Na_V1.4 are an established cause of hypokalemic periodic paralysis, an inherited disorder of skeletal muscle involving recurrent episodes of weakness in conjunction with low serum K⁺. Expression studies in oocytes have revealed anomalous, hyperpolarization-activated gating pore currents in mutant channels. This aberrant gating pore conductance creates a small inward current at the resting potential that is thought to contribute to susceptibility to depolarization in low K⁺ during attacks of weakness. A critical component of this hypothesis is the magnitude of the gating pore conductance relative to other conductances that are active at the resting potential in mammalian muscle: large enough to favor episodes of paradoxical depolarization in low K⁺, yet not so large as to permanently depolarize the fiber. To improve the estimate of the specific conductance for the gating pore in affected muscle, we sequentially measured Na⁺ current through the channel pore, gating pore current, and gating charge displacement in oocytes expressing R669H, R672G, or wild-type Na_V1.4 channels. The relative conductance of the gating pore to that of the pore domain pathway for Na⁺ was 0.03%, which implies a specific conductance in muscle from heterozygous patients of ∼10 µS/cm² or 1% of the total resting conductance.Unexpectedly, our data also revealed a substantial decoupling between gating charge displacement and peak Na⁺ current for both R669H and R672G mutant channels. This decoupling predicts a reduced Na⁺ current density in affected muscle, consistent with the observations that the maximal dV/dt and peak amplitude of the action potential are reduced in fibers from patients with R672G and in a knock-in mouse model of R669H. The defective coupling between gating charge displacement and channel activation identifies a previously unappreciated mechanism that contributes to the reduced excitability of affected fibers seen with these mutations and possibly with other R/X mutations of S4 of Na_V, Ca_V, and K_V channels associated with human disease.     

A CaVbeta SH3/guanylate kinase domain interaction regulates multiple properties of voltage-gated Ca2+ channels

Auxiliary Ca²⁺ channel β subunits (Ca_Vβ) regulate cellular Ca²⁺ signaling by trafficking pore-forming α₁ subunits to the membrane and normalizing channel gating. These effects are mediated through a characteristic src homology 3/guanylate kinase (SH3–GK) structural module, a design feature shared in common with the membrane-associated guanylate kinase (MAGUK) family of scaffold proteins. However, the mechanisms by which the Ca_Vβ SH3–GK module regulates multiple Ca²⁺ channel functions are not well understood. Here, using a split-domain approach, we investigated the role of the interrelationship between Ca_Vβ SH3 and GK domains in defining channel properties. The studies build upon a previously identified split-domain pair that displays a trans SH3–GK interaction, and fully reconstitutes Ca_Vβ effects on channel trafficking, activation gating, and increased open probability (P_o). Here, by varying the precise locations used to separate SH3 and GK domains and monitoring subsequent SH3–GK interactions by fluorescence resonance energy transfer (FRET), we identified a particular split-domain pair that displayed a subtly altered configuration of the trans SH3–GK interaction. Remarkably, this pair discriminated between Ca_Vβ trafficking and gating properties: α_1C targeting to the membrane was fully reconstituted, whereas shifts in activation gating and increased P_o functions were selectively lost. A more extreme case, in which the trans SH3–GK interaction was selectively ablated, yielded a split-domain pair that could reconstitute neither the trafficking nor gating-modulation functions, even though both moieties could independently engage their respective binding sites on the α_1C (Ca_V1.2) subunit. The results reveal that Ca_Vβ SH3 and GK domains function codependently to tune Ca²⁺ channel trafficking and gating properties, and suggest new paradigms for physiological and therapeutic regulation of Ca²⁺ channel activity.