Structural determinants of L-type channel activation in segment IIS6 revealed by a retinal disorder

The mechanism of channel opening for voltage-gated calcium channels is poorly understood. The importance of a conserved isoleucine residue in the pore-lining segment IIS6 has recently been highlighted by functional analyses of a mutation (I745T) in the Ca(V)1.4 channel causing severe visual impairment (Hemara-Wahanui, A., Berjukow, S., Hope, C. I., Dearden, P. K., Wu, S. B., Wilson-Wheeler, J., Sharp, D. M., Lundon-Treweek, P., Clover, G. M., Hoda, J. C., Striessnig, J., Marksteiner, R., Hering, S., and Maw, M. A. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7553-7558). In the present study we analyzed the influence of amino acids in segment IIS6 on gating of the Ca(V)1.2 channel. Substitution of Ile-781, the Ca(V)1.2 residue corresponding to Ile-745 in Ca(V)1.4, by residues of different hydrophobicity, size and polarity shifted channel activation in the hyperpolarizing direction (I781P > I781T > I781N > I781A > I781L). As I781P caused the most dramatic shift (-37 mV), substitution with this amino acid was used to probe the role of other residues in IIS6 in the process of channel activation. Mutations revealed a high correlation between the midpoint voltages of activation and inactivation. A unique kinetic phenotype was observed for residues 779-782 (LAIA) located in the lower third of segment IIS6; a shift in the voltage dependence of activation was accompanied by a deceleration of activation at hyperpolarized potentials, a deceleration of deactivation at all potentials (I781P and I781T), and decreased inactivation. These findings indicate that Ile-781 substitutions both destabilize the closed conformation and stabilize the open conformation of Ca(V)1.2. Moreover there may be a flexible center of helix bending at positions 779-782 of Ca(V)1.2. These four residues are completely conserved in high voltage-activated calcium channels suggesting that these channels may share a common mechanism of gating.     

Molecular modeling of benzothiazepine binding in the L-type calcium channel

Benz(othi)azepine (BTZ) derivatives constitute one of three major classes of L-type Ca(2+) channel ligands. Despite intensive experimental studies, no three-dimensional model of BTZ binding is available. Here we have built KvAP- and KcsA-based models of the Ca(v)1.2 pore domain in the open and closed states and used multiple Monte Carlo minimizations to dock representative ligands. In our open channel model, key functional groups of BTZs interact with BTZ-sensing residues, which were identified in previous mutational experiments. The bulky tricyclic moiety occupies interface between domains III and IV, while the ammonium group protrudes into the inner pore, where it is stabilized by nucleophilic C-ends of the pore helices. In the closed channel model, contacts with several ligand-sensing residues in the inner helices are lost, which weakens ligand-channel interactions. An important feature of the ligand-binding mode in both open and closed channels is an interaction between the BTZ carbonyl group and a Ca(2+) ion chelated by the selectivity filter glutamates in domains III and IV. In the absence of Ca(2+), the tricyclic BTZ moiety remains in the domain interface, while the ammonium group directly interacts with a glutamate residue in the selectivity filter. Our model suggests that the Ca(2+) potentiation involves a direct electrostatic interaction between aCa(2+) ion and the ligand rather than an allosteric mechanism. Energy profiles indicate that BTZs can reach the binding site from the domain interface, whereas access through the open activation gate is unlikely, because reorientation of the bulky molecule in the pore is hindered.     

Distinct mechanisms of block of Kv1.5 channels by tertiary and quaternary amine clofilium compounds

The quaternary ammonium compound clofilium and its tertiary amine derivative LY97241 were used to analyze mechanisms of block in a voltage-gated potassium channel. Wild-type and mutant Kv1.5 channels expressed in Xenopus oocytes were recorded by two-electrode voltage clamp. Open-channel block to 20% of the control current amplitude was induced reversibly by 50 microM clofilium or 200 microM LY97241, and was seen as an acceleration of the macroscopic current decay. Although blockers remained present after application, channels recovered from block during each interpulse interval. The optimum voltage for recovery (-45 mV at pH 7.3) at the threshold for channel activation indicated that clofilium block and recovery occurred principally through the open channel state. In contrast, LY97241 appeared to exit from the closed state and the open state. In an acid-tolerant Kv1.5 mutant channel (H452Q), external pH was used to titrate LY97241. At low pH, which protonates the LY97241 amine group, recovery from block at hyperpolarized potentials was impaired in a manner similar to that seen with clofilium. Recovery from clofilium block was reduced at negative potentials independent of pH, an effect attributed to trapping of the permanently charged compound within the closed channels.     

Bastadin 10 stabilizes the open conformation of the ryanodine-sensitive Ca(2+) channel in an FKBP12-dependent manner.

The marine sponge Ianthella basta synthesizes at least 25 tetrameric bromotyrosine structures that possess a stringent structural requirement for modifying the gating behavior of ryanodine-sensitive Ca(2+) channels (ryanodine receptors) (RyR)). Bastadin 5 (B5) was shown to stabilize open and closed channel states with little influence on the sensitivity of the channel to activation by Ca(2+) (Mack, M. M., Molinski, T. F., Buck, E. D., and Pessah, I. N. (1994) J. Biol. Chem. 269, 23236-23249). In the present paper, we utilize single channel analysis and measurements of Ca(2+) flux across the sarcoplasmic reticulum to identify bastadin 10 (B10) as the structural congener responsible for dramatically stabilizing the open conformation of the RyR channel, possibly by reducing the free energy associated with closed to open channel transitions (DeltaG*c --> o). The stability of the channel open state induced by B10 sensitized the channel to activation by Ca(2+) to such an extent that it essentially obviated regulation by physiological concentrations of Ca(2+) and relieved inhibition by physiological Mg(2+). These actions of B10 were produced only on the cytoplasmic face of the channel, were selectively eliminated by pretreatment of channels with FK506 or rapamycin, and were reconstituted by human recombinant FKBP12. The actions of B10 were found to be reversible. A structure-activity model is proposed by which substitutions on the Eastern and Western hemispheres of the bastarane macrocycle may confer specificity toward the RyR1-FKBP12 complex to stabilize either the closed or open channel conformation. These results indicate that RyR1-FKBP12 complexes possesses a novel binding domain for phenoxycatechols and raise the possibility of molecular recognition of an endogenous ligand.     

Calmodulin kinase II is involved in voltage-dependent facilitation of the L-type Cav1.2 calcium channel: Identification of the phosphorylation sites

Calcium-dependent facilitation of L-type calcium channels has been reported to depend on the function of calmodulin kinase II. In contrast, the mechanism for voltage-dependent facilitation is not clear. In HEK 293 cells expressing Ca(v)1.2, Ca(v)beta2a, and calmodulin kinase II, the calcium current measured at +30 mV was facilitated up to 1.5-fold by a 200-ms-long prepulse to +160 mV. This voltage-dependent facilitation was prevented by the calmodulin kinase II inhibitors KN93 and the autocamtide-2-related peptide. In cells expressing the Ca(v)1.2 mutation I1649E, a residue critical for the binding of Ca2+-bound calmodulin, facilitation was also abolished. Calmodulin kinase II was coimmunoprecipitated with the Ca(v)1.2 channel from murine heart and HEK 293 cells expressing Ca(v)1.2 and calmodulinkinase II. The precipitated Ca(v)1.2 channel was phosphorylated in the presence of calmodulin and Ca2+. Fifteen putative calmodulin kinase II phosphorylation sites were identified mostly in the carboxyl-terminal tail of Ca(v)1.2. Neither truncation at amino acid 1728 nor changing the II-III loop serines 808 and 888 to alanines affected facilitation of the calcium current. In contrast, facilitation was decreased by the single mutations S1512A and S1570A and abolished by the double mutation S1512A/S1570A. These serines flank the carboxyl-terminal EF-hand motif. Immunoprecipitation of calmodulin kinase II with the Ca(v)1.2 channel was not affected by the mutation S1512A/S1570A. The phosphorylation of the Ca(v)1.2 protein was strongly decreased in the S1512A/S1570A double mutant. These results suggest that voltage-dependent facilitation of the Ca(v)1.2 channel depends on the phosphorylation of Ser1512/Ser1570 by calmodulin kinase II.     

Are Cav1.3 pacemaker channels in chromaffin cells?: Possible bias from resting cell conditions and DHP blocker usage

Mouse and rat chromaffin cells (MCCs, RCCs) fire spontaneously at rest and their activity is mainly supported by the two L-type Ca(2+) channels expressed in these cells (Ca(v)1.2 and Ca(v)1.3). Using Ca(v)1.3(-/-) KO MCCs we have shown that Ca(v)1.3 possess all the prerequisites for carrying subthreshold currents that sustain low frequency cell firing near resting (0.5 to 2 Hz at -50 mV): low-threshold and steep voltage dependence of activation, slow and incomplete inactivation during pulses of several hundreds of milliseconds. Ca(v)1.2 contributes also to pacemaking MCCs and possibly even Na(+) channels may participate in the firing of a small percentage of cells. We now show that at potentials near resting (-50 mV), Ca(v)1.3 carries equal amounts of Ca(2+) current to Ca(v)1.2 but activates at 9 mV more negative potentials. MCCs express only TTX-sensitive Na(v)1 channels that activate at 24 mV more positive potentials than Ca(v)1.3 and are fully inactivating. Their blockade prevents the firing only in a small percentage of cells (13%). This suggests that the order of importance with regard to pacemaking MCCs is: Ca(v)1.3, Ca(v)1.2 and Na(v)1. The above conclusions, however, rely on the proper use of DHPs, whose blocking potency is strongly holding potential dependent. We also show that small increases of KCl concentration steadily depolarize the MCCs causing abnormally increased firing frequencies, lowered and broadened AP waveforms and an increased facility of switching "non-firing" into "firing" cells that may lead to erroneous conclusions about the role of Ca(v)1.3 and Ca(v)1.2 as pacemaker channels in MCCs.     

The saxitoxin/tetrodotoxin binding site on cloned rat brain IIa Na channels is in the transmembrane electric field.

The rat brain IIa (BrIIa) Na channel alpha-subunit and the brain beta 1 subunit were coexpressed in Xenopus oocytes, and peak whole-oocyte Na current (INa) was measured at a test potential of -10 mV. Hyperpolarization of the holding potential resulted in an increased affinity of STX and TTX rested-state block of BrIIa Na channels. The apparent half-block concentration (ED50) for STX of BrIIa current decreased with hyperpolarizing holding potentials (Vhold). At Vhold of -100 mV, the ED50 was 2.1 +/- 0.4 nM, and the affinity increased to a ED50 of 1.2 +/- 0.2 nM with Vhold of -140 mV. In the absence of toxin, the peak current amplitude was the same for all potentials negative to -90 mV, demonstrating that all of the channels were in a closed conformation and maximally available to open in this range of holding potentials. The Woodhull model (1973) was used to describe the increase of the STX ED50 as a function of holding potential. The equivalent electrical distance of block (delta) by STX was 0.18 from the extracellular milieu when the valence of STX was fixed to +2. Analysis of the holding potential dependence of TTX block yielded a similar delta when the valence of TTX was fixed to +1. We conclude that the guanidinium toxin site is located partially within the transmembrane electric field. Previous site-directed mutagenesis studies demonstrated that an isoform-specific phenylalanine in the BrIIa channel is critical for high affinity toxin block.(ABSTRACT TRUNCATED AT 250 WORDS)     

A smooth muscle Cav1.2 calcium channel splice variant underlies hyperpolarized window current and enhanced state-dependent inhibition by nifedipine

Native smooth muscle L-type Ca(v)1.2 calcium channels have been shown to support a fraction of Ca(2+) currents with a window current that is close to resting potential. The smooth muscle L-type Ca(2+) channels are also more susceptible to inhibition by dihydropyridines (DHPs) than the cardiac channels. It was hypothesized that smooth muscle Ca(v)1.2 channels exhibiting hyperpolarized shift in steady-state inactivation would contribute to larger inhibition by DHP, in addition to structural differences of the channels generated by alternative splicing that modulate DHP sensitivities. In addition, it has also been shown that alternative splicing modulates DHP sensitivities by generating structural differences in the Ca(v)1.2 channels. Here, we report a smooth muscle L-type Ca(v)1.2 calcium channel splice variant, Ca(v)1.2SM (1/8/9(*)/32/Delta33), that when expressed in HEK 293 cells display hyperpolarized shifts for steady-state inactivation and activation potentials when compared with the established Ca(v)1.2b clone (1/8/9(*)/32/33). This variant activates from more negative potentials and generates a window current closer to resting membrane potential. We also identified the predominant cardiac isoform Ca(v)1.2CM clone (1a/8a/Delta9(*)/32/33) that is different from the established Ca(v)1.2a (1a/8a/Delta9(*)/31/33). Importantly, Ca(v)1.2SM channels were shown to be more sensitive to nifedipine blockade than Ca(v)1.2b and cardiac Ca(v)1.2CM channels when currents were recorded in either 5 mM Ba(2+) or 1.8 mM Ca(2+) external solutions. This is the first time that a smooth muscle Ca(v)1.2 splice variant has been identified functionally to possess biophysical property that can be linked to enhanced state-dependent block by DHP.     

Gating competence of constitutively open CLC-0 mutants revealed by the interaction with a small organic Inhibitor

Opening of CLC chloride channels is coupled to the translocation of the permeant anion. From the recent structure determination of bacterial CLC proteins in the closed and open configuration, a glutamate residue was hypothesized to form part of the Cl--sensitive gate. The negatively charged side-chain of the glutamate was suggested to occlude the permeation pathway in the closed state, while opening of a single protopore of the double-pore channel would reflect mainly a movement of this side-chain toward the extracellular pore vestibule, with little rearrangement of the rest of the channel. Here we show that mutating this critical residue (Glu166) in the prototype Torpedo CLC-0 to alanine, serine, or lysine leads to constitutively open channels, whereas a mutation to aspartate strongly slowed down opening. Furthermore, we investigated the interaction of the small organic channel blocker p-chlorophenoxy-acetic acid (CPA) with the mutants E166A and E166S. Both mutants were strongly inhibited by CPA at negative voltages with a >200-fold larger affinity than for wild-type CLC-0 (apparent KD at -140 mV approximately 4 micro M). A three-state linear model with an open state, a low-affinity and a high-affinity CPA-bound state can quantitatively describe steady-state and kinetic properties of the CPA block. The parameters of the model and additional mutagenesis suggest that the high-affinity CPA-bound state is similar to the closed configuration of the protopore gate of wild-type CLC-0. In the E166A mutant the glutamate side chain that occludes the permeation pathway is absent. Thus, if gating consists only in movement of this side-chain the mutant E166A should not be able to assume a closed conformation. It may thus be that fast gating in CLC-0 is more complex than anticipated from the bacterial structures.     

Cav1.4 encodes a calcium channel with low open probability and unitary conductance

When transiently expressed in tsA-201 cells, Ca(v)1.4 calcium channels support only modest whole-cell currents with unusually slow voltage-dependent inactivation kinetics. To examine the basis for this unique behavior we used cell-attached patch single-channel recordings using 100 mM external barium as the charge carrier to determine the single-channel properties of Ca(v)1.4 and to compare them to those of the Ca(v)1.2. Ca(v)1.4 channel openings occurred infrequently and were of brief duration. Moreover, openings occurred throughout the duration of the test depolarization, indicating that the slow inactivation kinetics observed at the whole-cell level are caused by sustained channel activity. Ca(v)1.4 and Ca(v)1.2 channels displayed similar latencies to first opening. Because of the rare occurrence of events, the probability of opening could not be precisely determined but was estimated to be <0.015 over a voltage range of -20 to +20 mV. The single-channel conductance of Ca(v)1.4 channels was approximately 4 pS compared with approximately 20 pS for Ca(v)1.2 under the same experimental conditions. Additionally, in the absence of divalent cations, Ca(v)1.4 channels pass cesium ions with a single-channel conductance of approximately 21 pS. Although Ca(v)1.2 opening events were best described kinetically with two open time constants, Ca(v)1.4 open times were best described by a single time constant. BayK8644 slightly enhanced the single-channel conductance in addition to increasing the open time constant for Ca(v)1.4 channels by approximately 45% without, however, causing the appearance of an additional slower gating mode. Overall, our data indicate that single Ca(v)1.4 channels support only minute amounts of calcium entry, suggesting that large numbers of these channels are needed to allow for significant whole-cell current activity, and providing a mechanism to reduce noise in the visual system.     

Functional properties of a newly identified C-terminal splice variant of Cav1.3 L-type Ca2+ channels

An intramolecular interaction between a distal (DCRD) and a proximal regulatory domain (PCRD) within the C terminus of long Ca(v)1.3 L-type Ca(2+) channels (Ca(v)1.3(L)) is a major determinant of their voltage- and Ca(2+)-dependent gating kinetics. Removal of these regulatory domains by alternative splicing generates Ca(v)1.3(42A) channels that activate at a more negative voltage range and exhibit more pronounced Ca(2+)-dependent inactivation. Here we describe the discovery of a novel short splice variant (Ca(v)1.3(43S)) that is expressed at high levels in the brain but not in the heart. It lacks the DCRD but, in contrast to Ca(v)1.3(42A), still contains PCRD. When expressed together with α2δ1 and β3 subunits in tsA-201 cells, Ca(v)1.3(43S) also activated at more negative voltages like Ca(v)1.3(42A) but Ca(2+)-dependent inactivation was less pronounced. Single channel recordings revealed much higher channel open probabilities for both short splice variants as compared with Ca(v)1.3(L). The presence of the proximal C terminus in Ca(v)1.3(43S) channels preserved their modulation by distal C terminus-containing Ca(v)1.3- and Ca(v)1.2-derived C-terminal peptides. Removal of the C-terminal modulation by alternative splicing also induced a faster decay of Ca(2+) influx during electrical activities mimicking trains of neuronal action potentials. Our findings extend the spectrum of functionally diverse Ca(v)1.3 L-type channels produced by tissue-specific alternative splicing. This diversity may help to fine tune Ca(2+) channel signaling and, in the case of short variants lacking a functional C-terminal modulation, prevent excessive Ca(2+) accumulation during burst firing in neurons. This may be especially important in neurons that are affected by Ca(2+)-induced neurodegenerative processes.     

Voltage-dependent calcium channels

Voltage-activated calcium channels can be divided into two subgroups based on their activation threshold, low-voltage-activated (LVA) and high-voltage-activated (HVA). Auxiliary subunits of the HVA calcium channels contribute significantly to biophysical properties of the channels. We have cloned and characterized members of two families of auxiliary subunits: alpha2delta and gamma. Two new alpha2delta subunits, alpha2delta-2 and alpha2delta-3, regulate all classes of HVA calcium channels. While the ubiquitous alpha2delta-2 modulates both neuronal and non-neuronal channels with similar efficiency, the alpha2delta-3 subunit regulates Ca(v)2.3 channels more effectively. Furthermore, alpha2delta-2 may modulate the LVA Ca(v)3.1 channel. Four new gamma subunits, gamma-2, gamma-3, gamma-4 and gamma-5, were characterized. The gamma-2 subunit modulated both the non-neuronal Ca(v)1.2 channel and the neuronal Ca(v)2.1 channel. The gamma-4 subunit affected only the Ca(v)2.1 channel. The gamma-5 subunit may be a regulatory subunit of the LVA Ca(v)3.1 channel. The Ca(v)1.2 channel is a major target for treatment of cardiovascular diseases. We have mapped the interaction site for clinically important channel blockers - dihydropyridines (DHPs) - and analysed the underlying inhibition mechanism. High-affinity inhibition is characterized by interaction with inactivated state of the channel. Its structural determinants are amino acids of the IVS6 segment, with smaller contribution of the IS6 segment, which contributes to voltage-dependence of DHP inhibition. Removal of amino acids responsible for the high-affinity inhibition revealed a low-affinity open channel block, in which amino acids of the IIIS5 and IIIS6 segments take part. Experiments with a permanently charged DHP suggested that there is another low-affinity interaction site on the alpha(1) subunit. We have cloned and characterized murine neuronal LVA Ca(v)3.1 channel. The channel has high sensitivity to the organic blocker mibefradil, moderate sensitivity to phenytoin, and low sensitivity to ethosuximide, amiloride and valproat. The channel is insensitive to tetrodotoxin and DHPs. The inorganic blockers Ni2+ and Cd2+ are moderately effective compared to La3+. The current through the Ca(v)3.1 channel inactivates faster with Ba2+ compared to Ca2+. Molecular determinants of fast inactivation are located in amino side of the intracellular carboxy terminus. The voltage dependence of charge movement is very shallow compared to the voltage dependence of current activation. Transfer of 30 % of charge correlates with activation of 70 % of measurable macroscopic current. Prolonged depolarization does not immobilize charge movement of the Ca(v)3.1 channel.     

Evidence for a deep pore activation gate in small conductance Ca2+-activated K+ channels

Small conductance calcium-gated potassium (SK) channels share an overall topology with voltage-gated potassium (K(v)) channels, but are distinct in that they are gated solely by calcium (Ca(2+)), not voltage. For K(v) channels there is strong evidence for an activation gate at the intracellular end of the pore, which was not revealed by substituted cysteine accessibility of the homologous region in SK2 channels. In this study, the divalent ions cadmium (Cd(2+)) and barium (Ba(2+)), and 2-aminoethyl methanethiosulfonate (MTSEA) were used to probe three sites in the SK2 channel pore, each intracellular to (on the selectivity filter side of) the region that forms the intracellular activation gate of voltage-gated ion channels. We report that Cd(2+) applied to the intracellular side of the membrane can modify a cysteine introduced to a site (V391C) just intracellular to the putative activation gate whether channels are open or closed. Similarly, MTSEA applied to the intracellular side of the membrane can access a cysteine residue (A384C) that, based on homology to potassium (K) channel crystal structures (i.e., the KcsA/MthK model), resides one amino acid intracellular to the glycine gating hinge. Cd(2+) and MTSEA modify with similar rates whether the channels are open or closed. In contrast, Ba(2+) applied to the intracellular side of the membrane, which is believed to block at the intracellular end of the selectivity filter, blocks open but not closed channels when applied to the cytoplasmic face of rSK2 channels. Moreover, Ba(2+) is trapped in SK2 channels when applied to open channels that are subsequently closed. Ba(2+) pre-block slows MTSEA modification of A384C in open but not in closed (Ba(2+)-trapped) channels. The findings suggest that the SK channel activation gate resides deep in the vestibule of the channel, perhaps in the selectivity filter itself.     

The calcium-independent transient outward potassium current in isolated ferret right ventricular myocytes. II. Closed state reverse use- dependent block by 4-aminopyridine

Block of the calcium-independent transient outward K+ current, I(to), by 4-aminopyridine (4-AP) was studied in ferret right ventricular myocytes using the whole cell patch clamp technique. 4-AP reduces I(to) through a closed state blocking mechanism displaying "reverse use- dependent" behavior that was inferred from: (a) development of tonic block at hyperpolarized potentials; (b) inhibition of development of tonic block at depolarized potentials; (c) appearance of "crossover phenomena" in which the peak current is delayed in the presence of 4-AP at depolarized potentials; (d) relief of block at depolarized potentials which is concentration dependent and parallels steady-state inactivation for low 4-AP concentrations (V1/2 approximately -10 mV in 0.1 mM 4-AP) and steady-state activation at higher concentrations (V1/2 = +7 mV in 1 mM 4-AP, +15 mV in 10 mM 4-AP); and (e) reassociation of 4- AP at hyperpolarized potentials. No evidence for interaction of 4-AP with either the open or inactivated state of the I(to) channel was obtained from measurements of kinetics of recovery and deactivation in the presence of 0.5-1.0 mM 4-AP. At hyperpolarized potentials (-30 to - 90 mV) 10 mM 4-AP associates slowly (time constants ranging from approximately 800 to 1,300 ms) with the closed states of the channel (apparent Kd approximately 0.2 mM). From -90 to -20 mV the affinity of the I(to) channel for 4-AP appears to be voltage insensitive; however, at depolarized potentials (+20 to +100 mV) 4-AP dissociates with time constants ranging from approximately 350 to 150 ms. Consequently, the properties of 4-AP binding to the I(to) channel undergo a transition in the range of potentials over which channel activation and inactivation occurs (-30 to +20 mV). We propose a closed state model of I(to) channel gating and 4-AP binding kinetics, in which 4-AP binds to three closed states. In this model 4-AP has a progressively lower affinity as the channel approaches the open state, but has no intrinsic voltage dependence of binding.     

Molecular dynamics of a thermostable multicopper oxidase from Thermus thermophilus HB27: structural differences between the apo and holo forms

Molecular dynamic (MD) simulations have been performed on Tth-MCO, a hyperthermophilic multicopper oxidase from thermus thermophilus HB27, in the apo as well as the holo form, with the aim of exploring the structural dynamic properties common to the two conformational states. According to structural comparison between this enzyme and other MCOs, the substrate in process to electron transfer in an outer-sphere event seems to transiently occupy a shallow and overall hydrophobic cavity near the Cu type 1 (T1Cu). The linker connecting the β-strands 21 and 24 of the second domain (loop (β21-β24)(D2)) has the same conformation in both states, forming a flexible lid at the entrance of the electron-transfer cavity. Loop (β21-β24)(D2) has been tentatively assigned a role occluding the access to the electron-transfer site. The dynamic of the loop (β21-β24)(D2) has been investigated by MD simulation, and results show that the structures of both species have the same secondary and tertiary structure during almost all the MD simulations. In the simulation, loop (β21-β24)(D2) of the holo form undergoes a higher mobility than in the apo form. In fact, loop (β21-β24)(D2) of the holo form experiences a conformational change which enables exposure to the electron-transfer site (open conformation), while in the apo form the opposite effect takes place (closed conformation). To confirm the hypothesis that the open conformation might facilitate the transient electron-donor molecule occupation of the site, the simulation was extended another 40 ns with the electron-donor molecule docked into the protein cavity. Upon electron-donor molecule stabilization, loops near the cavity reduce their mobility. These findings show that coordination between the copper and the protein might play an important role in the general mobility of the enzyme, and that the open conformation seems to be required for the electron transfer process to T1Cu.     

Myocytes isolated from porcine coronary arteries: reduction of currents through L-type Ca-channels by verapamil-type Ca-antagonists.

Myocytes were enzymatically isolated from large epicardial arteries of the pig. In the cell attached configuration, we studied currents through L-type Ca-channels. At 22 degrees C, open channel conductance was 9 pS with 110 mM Ca2+ and 24 pS with 110 mM Ba2+ as charge carrier. According to the life time of the open state, 2 'modes' of gating are distinguished; mode 1 contributed time constants shorter than 1 ms, mode 2 those longer than 6 ms to the open time distribution. Mode 2 openings appeared spontaneously, more frequently with Ba2+ than with Ca2+ as charge carrier. The Ca-agonist Bay K 8644 (0.5 microM) facilitated the appearance of mode 2. Bath application of the phenylalkylamine D600 (1 microM) did not change the gating modes, but it reduced the channel openness by increasing the percentage of blank records. With whole cell recordings, we studied reduction of ICa by 1 microM D 600 at 3.6 mM [Ca2+] and 35 degrees C. At a holding potential of -45 mV, D 600 induced an 'initial block' of 35% (10% at -65 mV). Upon repetitive 1 Hz pulsing (170 ms to 0 mV) an additional, 'use-dependent' block developed with time. More negative holding potentials attenuated reduction of ICa by D 600, hyperpolarizations to -100 mV had an 'unblocking' effect. In regard to reduction of ICa, we compared the partially uncharged D 600 (membrane permeable) with the completely charged compound D 890 (membrane impermeable). When applied with the bath, 1 or 10 microM D 600 reduced ICa dose-dependently whereas D 890 was ineffective. When D 890 was applied via the patch electrode to the cytosol, it reduced ICa. We discuss that D 600 enters the cell in the uncharged lipid soluble form and reaches form the inside its receptor associated with the Ca-channel.     

Paxilline inhibits BK channels by an almost exclusively closed-channel block mechanism

Paxilline, a tremorogenic fungal alkaloid, potently inhibits large conductance Ca²⁺- and voltage-activated K⁺ (BK)-type channels, but little is known about the mechanism underlying this inhibition. Here we show that inhibition is inversely dependent on BK channel open probability (Po), and is fully relieved by conditions that increase Po, even in the constant presence of paxilline. Manipulations that shift BK gating to more negative potentials reduce inhibition by paxilline in accordance with the increase in channel Po. Measurements of Po times the number of channels at negative potentials support the idea that paxilline increases occupancy of closed states, effectively reducing the closed–open equilibrium constant, L(0). Gating current measurements exclude an effect of paxilline on voltage sensors. Steady-state inhibition by multiple paxilline concentrations was determined for four distinct equilibration conditions, each with a distinct Po. The IC₅₀ for paxilline shifted from around 10 nM when channels were largely closed to near 10 µM as maximal Po was approached. Model-dependent analysis suggests a mechanism of inhibition in which binding of a single paxilline molecule allosterically alters the intrinsic L(0) favoring occupancy of closed states, with affinity for the closed conformation being >500-fold greater than affinity for the open conformation. The rate of inhibition of closed channels was linear up through 2 µM paxilline, with a slope of 2 × 10⁶ M⁻¹s⁻¹. Paxilline inhibition was hindered by either the bulky cytosolic blocker, bbTBA, or by concentrations of cytosolic sucrose that hinder ion permeation. However, paxilline does not hinder MTSET modification of the inner cavity residue, A313C. We conclude that paxilline binds more tightly to the closed conformation, favoring occupancy of closed-channel conformations, and propose that it binds to a superficial position near the entrance to the central cavity, but does not hinder access of smaller molecules to this cavity.     

Regulation of K+ flow by a ring of negative charges in the outer pore of BKCa channels. Part II: Neutralization of aspartate 292 reduces long channel openings and gating current slow component

Neutralization of the aspartate near the selectivity filter in the GYGD pore sequence (D292N) of the voltage- and Ca(2+)-activated K+ channel (MaxiK, BKCa) does not prevent conduction like the corresponding mutation in Shaker channel, but profoundly affects major biophysical properties of the channel (Haug, T., D. Sigg, S. Ciani, L. Toro, E. Stefani, and R. Olcese. 2004. J. Gen. Physiol. 124:173-184). Upon depolarizations, the D292N mutant elicited mostly gating current, followed by small or no ionic current, at voltages where the wild-type hSlo channel displayed robust ionic current. In fact, while the voltage dependence of the gating current was not significantly affected by the mutation, the overall activation curve was shifted by approximately 20 mV toward more depolarized potentials. Several lines of evidence suggest that the mutation prevents population of certain open states that in the wild type lead to high open probability. The activation curves of WT and D292N can both be fitted to the sum of two Boltzmann distributions with identical slope factors and half activation potentials, just by changing their relative amplitudes. The steeper and more negative component of the activation curve was drastically reduced by the D292N mutation (from 0.65 to 0.30), suggesting that the population of open states that occurs early in the activation pathway is reduced. Furthermore, the slow component of the gating current, which has been suggested to reflect transitions from closed to open states, was greatly reduced in D292N channels. The D292N mutation also affected the limiting open probability: at 0 mV, the limiting open probability dropped from approximately 0.5 for the wild-type channel to 0.06 in D292N (in 1 mM [Ca2+]i). In addition to these effects on gating charge and open probability, as already described in Part I, the D292N mutation introduces a approximately 40% reduction of outward single channel conductance, as well as a strong outward rectification.     

Mapping of dihydropyridine binding residues in a less sensitive invertebrate L-type calcium channel (LCa v 1)

Invertebrate L-type calcium channel, LCa(v) 1, isolated from the pond snail Lymnaea stagnalis is nearly indistinguishable from mammalian Ca(v) 1.2 (α1C) calcium channel in biophysical characteristics observed in vitro. These L-type channels are likely constrained within a narrow range of biophysical parameters to perform similar functions in the snail and mammalian cardiovascular systems. What distinguishes snail and mammalian L-type channels is a difference in dihydropyridine sensitivity: 100 nM isradipine exhibits a significant block of mammalian Ca(v) 1.2 currents without effect on snail LCa(v)1 currents. The native snail channel serves as a valuable surrogate for validating key residue differences identified from previous experimental and molecular modeling work. As predicted, three residue changes in LCa(v)1 (N_3o18, F_3i10, and I_4i12) replaced with DHP-sensing residues in respective positions of Ca(v) 1.2, (Q_3o18, Y_3i10, and M_4i12) raises the potency of isradipine block of LCa(v)1 channels to that of mammalian Ca(v) 1.2. Interestingly, the single N_3o18_Q mutation in LCa(v) 1 channels lowers DHP sensitivity even further and the triple mutation bearing enhanced isradipine sensitivity, still retains a reduced potency of agonist, (S)-Bay K8644.     

Calmodulin overexpression does not alter Cav1.2 function or oligomerization state

Interactions between calmodulin (CaM) and voltage-gated calcium channels (Ca(v)s) are crucial for Ca(v) activity-dependent feedback modulation. We recently reported an X-ray structure that shows two Ca(2+)/CaM molecules bound to the Ca(v)1.2 C terminal tail, one at the PreIQ region and one at the IQ domain. Surprisingly, the asymmetric unit of the crystal showed a dimer in which Ca(2+)/CaM bridged two PreIQ helixes to form a 4:2 Ca(2+)/CaM:Ca(v) C-terminal tail assembly. Contrary to previous proposals based on a similar crystallographic dimer, extensive biochemical analysis together with subunit counting experiments of full-length channels in live cell membranes failed to find evidence for multimers that would be compatible with the 4:2 crossbridged complex. Here, we examine this possibility further. We find that CaM over-expression has no functional effect on Ca(v)1.2 inactivation or on the stoichiometry of full-length Ca(v)1.2. These data provide further support for the monomeric Ca(v)1.2 stoichiometry. Analysis of the electrostatic surfaces of the 2:1 Ca(2+)/CaM:Ca(V) C-terminal tail assembly reveals notable patches of electronegativity. These could influence various forms of channel modulation by interacting with positively charged elements from other intracellular channel domains.