The ?2?-1 subunit remodels CaV1.2 voltage sensors and allows Ca2+ influx at physiological membrane potentials.
ABSTRACT: Excitation-evoked calcium influx across cellular membranes is strictly controlled by voltage-gated calcium channels (CaV), which possess four distinct voltage-sensing domains (VSDs) that direct the opening of a central pore. The energetic interactions between the VSDs and the pore are critical for tuning the channel's voltage dependence. The accessory ?2?-1 subunit is known to facilitate CaV1.2 voltage-dependent activation, but the underlying mechanism is unknown. In this study, using voltage clamp fluorometry, we track the activation of the four individual VSDs in a human L-type CaV1.2 channel consisting of ?1C and ?3 subunits. We find that, without ?2?-1, the channel complex displays a right-shifted voltage dependence such that currents mainly develop at nonphysiological membrane potentials because of very weak VSD-pore interactions. The presence of ?2?-1 facilitates channel activation by increasing the voltage sensitivity (i.e., the effective charge) of VSDs I-III. Moreover, the ?2?-1 subunit also makes VSDs I-III more efficient at opening the channel by increasing the coupling energy between VSDs II and III and the pore, thus allowing Ca influx within the range of physiological membrane potentials.
Project description:Excitation-evoked Ca(2+) influx is the fastest and most ubiquitous chemical trigger for cellular processes, including neurotransmitter release, muscle contraction, and gene expression. The voltage dependence and timing of Ca(2+) entry are thought to be functions of voltage-gated calcium (CaV) channels composed of a central pore regulated by four nonidentical voltage-sensing domains (VSDs I-IV). Currently, the individual voltage dependence and the contribution to pore opening of each VSD remain largely unknown. Using an optical approach (voltage-clamp fluorometry) to track the movement of the individual voltage sensors, we discovered that the four VSDs of CaV1.2 channels undergo voltage-evoked conformational rearrangements, each exhibiting distinct voltage- and time-dependent properties over a wide range of potentials and kinetics. The voltage dependence and fast kinetic components in the activation of VSDs II and III were compatible with the ionic current properties, suggesting that these voltage sensors are involved in CaV1.2 activation. This view is supported by an obligatory model, in which activation of VSDs II and III is necessary to open the pore. When these data were interpreted in view of an allosteric model, where pore opening is intrinsically independent but biased by VSD activation, VSDs II and III were each found to supply ?50 meV (?2 kT), amounting to ?85% of the total energy, toward stabilizing the open state, with a smaller contribution from VSD I (?16 meV). VSD IV did not appear to participate in channel opening.
Project description:Point mutations in pore-lining S6 segments of CaV1.2 shift the voltage dependence of activation into the hyperpolarizing direction and significantly decelerate current activation and deactivation. Here, we analyze theses changes in channel gating in terms of a circular four-state model accounting for an activation R-A-O and a deactivation O-D-R pathway. Transitions between resting-closed (R) and activated-closed (A) states (rate constants x(V) and y(V)) and open (O) and deactivated-open (D) states (u(V) and w(V)) describe voltage-dependent sensor movements. Voltage-independent pore openings and closures during activation (A-O) and deactivation (D-R) are described by rate constants alpha and beta, and gamma and delta, respectively. Rate constants were determined for 16-channel constructs assuming that pore mutations in IIS6 do not affect the activating transition of the voltage-sensing machinery (x(V) and y(V)). Estimated model parameters of 15 CaV1.2 constructs well describe the activation and deactivation processes. Voltage dependence of the "pore-releasing" sensor movement ((x(V)) was much weaker than the voltage dependence of "pore-locking" sensor movement (y(V)). Our data suggest that changes in membrane voltage are more efficient in closing than in opening CaV1.2. The model failed to reproduce current kinetics of mutation A780P that was, however, accurately fitted with individually adjusted x(V) and y(V). We speculate that structural changes induced by a proline substitution in this position may disturb the voltage-sensing domain.
Project description:Voltage sensors (VSs) initiate the pore opening and closure in voltage-gated ion channels. Here, we propose a technique for estimation of the equilibrium constant of the up- and downward VS movements and rate constants of pore transitions from macroscopic current kinetics. Bell-shaped voltage dependence of the activation/deactivation time constants and Bolzmann distributions of CaV1.2 activation were analyzed in terms of a circular four-state (rest, activated, open, deactivated) channel model: both dependencies uniquely constrain the model parameters. Neutralization of gating charges in IS4 or IIS4 only slightly affects the equilibrium constant of VS transition while affecting simultaneously the rate constants of pore opening and closure. The application of our technique revealed that pore mutations on IS6-IVS6 segments induce pronounced shifts of the VS equilibrium between the resting (down) and activated (up) position. Analyzing a channelopathy mutation highlighted that the leftward shift of the activation curve induced by I781T on IIS6 is only partially (35 %) caused by a destabilization of the channel pore but predominantly (65 %) by a shifted VS equilibrium towards activation. The algorithm proposed for CaV1.2 may be applicable for calculating rate constants from macroscopic current kinetics in other voltage-gated ion channels.
Project description:Voltage-dependent calcium channels (CaV) activate over a wide range of membrane potentials, and the voltage-dependence of activation of specific channel isoforms is exquisitely tuned to their diverse functions in excitable cells. Alternative splicing further adds to the stunning diversity of gating properties. For example, developmentally regulated insertion of an alternatively spliced exon 29 in the fourth voltage-sensing domain (VSD IV) of CaV1.1 right-shifts voltage-dependence of activation by 30 mV and decreases the current amplitude several-fold. Previously we demonstrated that this regulation of gating properties depends on interactions between positive gating charges (R1, R2) and a negative countercharge (D4) in VSD IV of CaV1.1. Here we investigated whether this molecular mechanism plays a similar role in the VSD IV of CaV1.3 and in VSDs II and IV of CaV1.2 by introducing charge-neutralizing mutations (D4N or E4Q) in the corresponding positions of CaV1.3 and in two splice variants of CaV1.2. In both channels the D4N (VSD IV) mutation resulted in a ?5 mV right-shift of the voltage-dependence of activation and in a reduction of current density to about half of that in controls. However in CaV1.2 the effects were independent of alternative splicing, indicating that the two modulatory processes operate by distinct mechanisms. Together with our previous findings these results suggest that molecular interactions engaging D4 in VSD IV contribute to voltage-sensing in all examined CaV1 channels, however its striking role in regulating the gating properties by alternative splicing appears to be a unique property of the skeletal muscle CaV1.1 channel.
Project description:Activity of voltage-gated Cav1.3 L-type Ca(2+) channels is required for proper hearing as well as sinoatrial node and brain function. This critically depends on their negative activation voltage range, which is further fine-tuned by alternative splicing. Shorter variants miss a C-terminal regulatory domain (CTM), which allows them to activate at even more negative potentials than C-terminally long-splice variants. It is at present unclear whether this is due to an increased voltage sensitivity of the Cav1.3 voltage-sensing domain, or an enhanced coupling of voltage-sensor conformational changes to the subsequent opening of the activation gate. We studied the voltage-dependence of voltage-sensor charge movement (QON-V) and of current activation (ICa-V) of the long (Cav1.3L) and a short Cav1.3 splice variant (Cav1.342A) expressed in tsA-201 cells using whole cell patch-clamp. Charge movement (QON) of Cav1.3L displayed a much steeper voltage-dependence and a more negative half-maximal activation voltage than Cav1.2 and Cav3.1. However, a significantly higher fraction of the total charge had to move for activation of Cav1.3 half-maximal conductance (Cav1.3: 68%; Cav1.2: 52%; Cav3.1: 22%). This indicated a weaker coupling of Cav1.3 voltage-sensor charge movement to pore opening. However, the coupling efficiency was strengthened in the absence of the CTM in Cav1.342A, thereby shifting ICa-V by 7.2 mV to potentials that were more negative without changing QON-V. We independently show that the presence of intracellular organic cations (such as n-methyl-D-glucamine) induces a pronounced negative shift of QON-V and a more negative activation of ICa-V of all three channels. These findings illustrate that the voltage sensors of Cav1.3 channels respond more sensitively to depolarization than those of Cav1.2 or Cav3.1. Weak coupling of voltage sensing to pore opening is enhanced in the absence of the CTM, allowing short Cav1.342A splice variants to activate at lower voltages without affecting QON-V.
Project description:Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a critical role in modulating the function of numerous ion channels, including large-conductance Ca(2+)- and voltage-dependent K(+) (BK, Slo1) channels. Slo1 BK channel complexes include four pore-forming Slo1 (?) subunits as well as various regulatory auxiliary subunits (? and ?) that are expressed in different tissues. We examined the molecular and biophysical mechanisms underlying the effects of brain-derived PIP2 on human Slo1 BK channel complexes with different subunit compositions that were heterologously expressed in human embryonic kidney cells. PIP2 inhibited macroscopic currents through Slo1 channels without auxiliary subunits and through Slo1 + ?1 complexes. In contrast, PIP2 markedly increased macroscopic currents through Slo1 + ?1 and Slo1 + ?4 channel complexes and failed to alter macroscopic currents through Slo1 + ?2 and Slo1 + ?2 ?2-19 channel complexes. Results obtained at various membrane potentials and divalent cation concentrations suggest that PIP2 promotes opening of the ion conduction gate in all channel types, regardless of the specific subunit composition. However, in the absence of ? subunits positioned near the voltage-sensor domains (VSDs), as in Slo1 and probably Slo1 + ?1, PIP2 augments the negative surface charge on the cytoplasmic side of the membrane, thereby shifting the voltage dependence of VSD-mediated activation in the positive direction. When ?1 or ?4 subunits occupy the space surrounding the VSDs, only the stimulatory effect of PIP2 is evident. The subunit compositions of native Slo1 BK channels differ in various cell types; thus, PIP2 may exert distinct tissue- and divalent cation-dependent modulatory influences.
Project description:Voltage-dependent potassium (Kv), sodium (Nav), and calcium channels open and close in response to changes in transmembrane (TM) potential, thus regulating cell excitability by controlling ion flow across the membrane. An outstanding question concerning voltage gating is how voltage-induced conformational changes of the channel voltage-sensing domains (VSDs) are coupled through the S4-S5 interfacial linking helices to the opening and closing of the pore domain (PD). To investigate the coupling between the VSDs and the PD, we generated a closed Kv channel configuration from Aeropyrum pernix (KvAP) using atomistic simulations with experiment-based restraints on the VSDs. Full closure of the channel required, in addition to the experimentally determined TM displacement, that the VSDs be displaced both inwardly and laterally around the PD. This twisting motion generates a tight hydrophobic interface between the S4-S5 linkers and the C-terminal ends of the pore domain S6 helices in agreement with available experimental evidence.
Project description:Allosteric interactions between the voltage-sensing domain (VSD), the Ca2+-binding sites, and the pore domain govern the mammalian Ca2+- and voltage-activated K+ (BK) channel opening. However, the functional relevance of the crosstalk between the Ca2+- and voltage-sensing mechanisms on BK channel gating is still debated. We examined the energetic interaction between Ca2+ binding and VSD activation by investigating the effects of internal Ca2+ on BK channel gating currents. Our results indicate that Ca2+ sensor occupancy has a strong impact on VSD activation through a coordinated interaction mechanism in which Ca2+ binding to a single ?-subunit affects all VSDs equally. Moreover, the two distinct high-affinity Ca2+-binding sites contained in the C-terminus domains, RCK1 and RCK2, contribute equally to decrease the free energy necessary to activate the VSD. We conclude that voltage-dependent gating and pore opening in BK channels is modulated to a great extent by the interaction between Ca2+ sensors and VSDs.
Project description:A classical voltage-gated ion channel consists of four voltage-sensing domains (VSDs). However, the roles of each VSD in the channels remain elusive. We developed a GVTDT (Graft VSD To Dimeric TASK3 channels that lack endogenous VSDs) strategy to produce voltage-gated channels with a reduced number of VSDs. TASK3 channels exhibit a high host tolerance to VSDs of various voltage-gated ion channels without interfering with the intrinsic properties of the TASK3 selectivity filter. The constructed channels, exemplified by the channels grafted with one or two VSDs from Kv7.1 channels, exhibit classical voltage sensitivity, including voltage-dependent opening and closing. Furthermore, the grafted Kv7.1 VSD transfers the potentiation activity of benzbromarone, an activator that acts on the VSDs of the donor channels, to the constructed channels. Our study indicates that one VSD is sufficient to voltage-dependently gate the pore and provides new insight into the roles of VSDs.
Project description:BACKGROUND: The L-type calcium channel Cav1.2 is important for brain and heart function. The ubiquitous calcium sensing protein calmodulin (CaM) regulates calcium dependent gating of Cav1.2 channels by reducing calcium influx, a process known as calcium-dependent inactivation (CDI). Dissecting the calcium-dependence of CaM in this process has benefited greatly from the use of mutant CaM molecules which are unable to bind calcium to their low affinity (N-lobe) and high affinity (C-lobe) binding sites. Unlike CDI, it is unknown whether CaM can modulate the activation gating of Cav1.2 channels. RESULTS: We examined a Cav1.2 point mutant in the N-terminus region of the channel (A39V) that has been previously linked to Brugada syndrome. Using mutant CaM constructs in which the N- and/or C-lobe calcium binding sites were ablated, we were able to show that this Brugada syndrome mutation disrupts N-lobe CDI of the channel. In the course of these experiments, we discovered that all mutant CaM molecules were able to alter the kinetics of channel activation even in the absence of calcium for WT-Cav1.2, but not A39V-Cav1.2 channels. Moreover, CaM mutants differentially shifted the voltage-dependence of activation for WT and A39V-Cav1.2 channels to hyperpolarized potentials. Our data therefore suggest that structural changes in CaM that arise directly from site directed mutagenesis of calcium binding domains alter activation gating of Cav1.2 channels independently of their effects on calcium binding, and that the N-terminus of the channel contributes to this CaM dependent process. CONCLUSIONS: Our data indicate that caution must be exercised when interpreting the effects of CaM mutants on ion channel gating.