Introduction

During an action potential calcium (Ca²⁺) ions enter the cell through voltage-gated Ca²⁺ channels (Ca_v). Ca_v channels first open and subsequently close before recovering to the resting state (fig. 1A). The process of channel closure during maintained membrane depolarization is called “inactivation”. During inactivation Ca_v channels undergo several conformational changes: one is mediated by intracellular Ca²⁺ interacting with a channel-calmodulin complex (Ca²⁺-dependent inactivation), two other conformational changes are voltage-dependent (fast and slow voltage-dependent inactivation, fig. 1A, see refs. 1, 2 for review, and other chapters in this book).

Figure 1

Ca²⁺ channel conformational states as potential drug targets. A) Voltage-gated Ca²⁺ (Ca_v) channels reside in closed (resting), activated (open) and several inactivated (I) states (i.e., Ca²⁺-dependent inactivation [I-Ca], fast [I-fast] and slow [I-slow] (more...)

Evolution has designed high-voltage-activated (HVA) Ca_v channel families (also known as L-type [Ca_v1.1, Ca_v1.2, Ca_v1.3, Ca_v1.4], P/Q-type [Ca_v2.1], N-type [Ca_v2.2] and R-type [Ca_v2.3] channels) and low-voltage-activated (LVA) Ca²⁺ channel family Ca_v3.³ The different family members display different inactivation properties.^1,2 HVA Ca_v channels are heterooligomeric protein complexes composed of an a1-subunit, auxiliary β-, α₂-δ- and in some channels of an additional γ-subunit.⁴ The α₁-subunits sense the membrane voltage, form the pore and define the basic pharmacological properties of the channels^5-8 (fig. 1C, D). Four β-subunit genes (β₁, β₂, β₃, β₄) and various splice variants have been identified. These subunits modulate the inactivation properties of HVA Ca_v (fig. 1B).⁶ HVA and LVA Ca_v channels undergo fast and slow voltage-dependent inactivation.^9-12 Ca²⁺/calmodulin-dependent inactivation is exclusively observed in HVA (Ca_v1.2, Ca_v2.1 and other HVA Ca_v channels).^13-17

The blocking action of many Ca²⁺ antagonists such as the 1,4-dihydropyridines (DHPs), phenylalkylamines (PAAs), diltiazem (DIL) and mibefradil (MIB) in myocardial, smooth muscle and neuronal cells is enhanced by membrane depolarizations.^18-23 This is conventionally explained by a higher affinity of these drugs for open or inactivated channels (fig. 1A). The precise mechanisms of channel block by these compounds remains to be elucidated.

Recent studies on chimeric and mutant Ca_v channels provided new insights into the underlying molecular events. In particular the design of channels with different inactivation properties may help us to understand the molecular role of inactivation in channel inhibition by Ca²⁺ antagonists.

Voltage-Dependent Inactivation and Drug Sensitivity of Chimeric Ca_v

First evidence for a link between structural determinants of Ca_v channel inactivation and drug-sensitivity came from studies on chimeric channels. In order to localise the drug-binding determinants for Ca²⁺ antagonists Grabner et al²⁴ inserted various sequence stretches of a PAA, DHP and DIL-sensitive α₁ subunit of an L-type channel into the poorly sensitive α₁2.1 (fig. 2A). Transfer of Ca_v1.2 segments IIIS5, IIIS6 and IVS6 to Ca_v2.1 confered PAA- and DIL-sensitivity to the resulting construct AL12 (fig.2B).²⁵

Figure 2

Inactivation properties of chimeric Ca_v affect drug sensitivity. Domains III and IV of wild type Ca_v2.1 and Ca_v2.1/Ca_v1 chimeric channels. Ca_v2.1 sequence is shown in white and L-type sequence as black transmembrane segments, corresponding barium currents (more...)

Analysis of the inactivation of some of the chimeric channels revealed a correlation between inactivation kinetics and drug sensitivity. Substitution of domains IIIS5, IIIS6 and the connecting linker (chimera AL20, fig. 2C) diminished both inactivation and block by DIL and PAA.²⁵ This was surprising because AL20 carries more than half of the PAA- and DIL-determinants (fig. 1D). The almost complete loss of drug sensitivity raised the suspicion that the process of channel inactivation might interfere with drug action. This hypothesis was further supported by chimera AL22 lacking all PAA- and DIL-determinants in segment IIIS6 but inactivating significantly faster than AL12 and Ca_v2.1 (fig. 2D). This channel exhibited PAA- and DIL-sensitivity comparable to (or even higher) than chimera AL12 and wild type Ca_v1.2.²⁵

A further example of a correlation between inactivation and drug-sensitivity in a chimeric channel comes from the work of Motoike et al²⁶ who substituted a small sequence stretch outside the putative drug binding region of Ca_v1.2 in the intracellular loop between IVS4 and IVS5 by the homologous residues from the rat brain Na⁺ channel (chimera HHT-5371). As illustrated in Figure 2E, this substitution simultaneously diminished channel inactivation and DIL-sensitivity.

Amino Acid Residues Located in the Putative Drug-Binding Region Affect Drug-Sensitivity and Channel Inactivation

Deeper insights into the interdependence between inactivation and channel block were enabled by the design of mutant Ca_v channels with different inactivation properties. Hering et al²⁵ demonstrated that transfer of only three Ca_v1.2-specific amino acids in segment IVS6 to Ca_v2.1 (Y1804, A1808 and I1811 a1A numbering, named chimera AL25²⁵, fig. 3A) confers high sensitivity for (-)gallopamil and DIL (fig. 3B, compare top left and top right traces). The authors paid, however, attention to the fact that these mutations significantly accelerated channel inactivation (fig. 3B, top left and right traces). Furthermore, mutating either A1808 back to serine or I1811 back to methionine (fig. 3B, middle panels) reduced not only PAA- and DIL-sensitivities but, simultaneously, the rate of channel inactivation.

Figure 3

Single amino acids in segment IVS6 of a diltiazem-sensitive Ca_v2.1 mutant affect drug sensitivity and channel inactivation. A) a-Helical representation of Ca_v2.1 segment IVS6. Transfer of four Ca_v1.2 amino acids (Y, A, I, M, highlighted as black circles, (more...)

On the other hand, insertion of a fourth Ca_v1.2 residue (M1805) into the corresponding position of AL25 further enhanced inactivation and sensitivity for (-)gallopamil and DIL (fig. 3B, bottom left). These data confirmed a close relation between the rate of intrinsic inactivation and the apparent sensitivity for Ca²⁺ antagonists. We hypothesise that the augmentation of the use-dependent block at least for some of the mutants is mediated by enhanced inactivation rather than by transfer of specific binding determinants.

Interestingly, mutation Y1804I decreased DIL- (and PAA-) sensitivity without significantly slowing the rate of channel inactivation (fig.3B bottom right). Y1804 is, therefore, more likely to contribute drug-binding energy than the other IVS6 residues that could equally modulate drug sensitivity in an indirect manner by affecting inactivation. A possible key role of Y1804 in drug binding is also supported by data from Degtiar et al who demonstrated low PAA-sensitivity of a chimeric construct (AL30) inactivating even faster than AL25 but lacking Y1804.²⁷

It was therefore interesting to study if the putative drug binding determinants in the drug sensitive wild type Ca_v1.2 would affect inactivation. A first analysis of the impact of IVS6 amino acids in equivalent positions (fig.4) revealed that substitution of I1470 (corresponding to the Ca_v2.1 M1811, (fig.3A) and the adjacent DHP-determinant I1471 by alanine both substantially slow channel inactivation (fig. 4A,B).

Figure 4

Inactivation determinants in Ca_v1.2 segment IVS6 modulate drug-sensitivity. A) a-Helical representation of Ca_v1.2 segment IVS6. Amino acid residues contributing to PAA- and DIL-sensitivity^, (see fig. 1D) are shown in black. A hot spot of inactivation (more...)

Drug-Sensitivity Is Affected by Inactivation Determinants Located Outside the Putative Drug-Binding Region

A similar correlation between channel inactivation and apparent sensitivity for Ca²⁺ antagonists was observed for amino acids substitutions outside the putative drug-binding region. As illustrated in Figure 4C, substitution of valine (V1478) in the lower part of segment IVS6 of Ca_v1.2 by alanine significantly reduced both inactivation and diltiazem sensitivity. A systematic analysis of amino acids in segment IIIS6 of a Ca_v1.1/Ca_v2.1 chimera revealed a strong correlation between the two parameters (fig. 5C).²⁸ Substitution of two residues in segment IIIS6 localised close to the inner mouth of the channel pore by alanine (I1163A and F1164A, fig. 5) and to an even greater extent the corresponding double mutation (IF1163,1164AA) gradually reduced the rate of current inactivation and simultaneously diminished use-dependent block by (-)gallopamil (fig. 5B).

Figure 5

Point mutations in Ca_v1.2 segment IIIS6 reveal correlation between inactivation and PAA-sensitivity. A) α-Helical representation of Ca_v1.2 segment IIIS6 forming part of the highly PAA sensitive Ca_v1.1/Ca_v2.1 chimera AL1. IIIS6 amino acid residues (more...)

By investigating the effect of a single amino acid substitution in the I-II linker (R387E in Ca_v2.1) Sokolov et al²⁹ observed a similar trend: the slower the channel inactivated the lower was the apparent sensitivity for (-)gallopamil. In addition, Jimenez et al³⁰ reported that ultraslow inactivation generated by a single valine insertion in to the I-II linker of the Ca_v1.2 α₁ subunit strongly reduced use-dependent channel block by MIB. Together these data illustrate that Ca²⁺ channel block is modulated by inactivation determinants that are located inside as well as outside of the putative drug-binding region.

β-Subunits Modulate Inactivation and Channel Inhibition

It is well established that the β-subunit composition of HVA Ca_v channels affects their inactivation properties.⁶ According to a widely accepted hypothesis many Ca²⁺ antagonists bind predominately (with high affinity) to the inactivated channel state.^20,21 Since β-subunits either enhance or reduce Ca_v channel inactivation, they have emerged as important tools for the analysis of state dependent drug action. Coexpression of the α₁ subunit with ‘accelerating’ β-subunits (β₁- or β₃-) was, therefore, expected to enhance channel block.

In line with this hypothesis Lacinova et al³¹ reported an almost 14-fold higher apparent sensitivity of Ca_v1.2 for the PAA verapamil if the Ca_v1.2 α₁-subunit was coexpressed with a β₃-subunit (Ca_v1.2(β₃)).

(-)Gallopamil inhibited Ca_v1.2(β₃) 3.4-fold stronger than Ca_v1.2 α₁ subunits expressed without β-subunits.³¹ Sokolov et al³² studied the underlying molecular mechanism and compared PAA-sensitivity of Ca_v1.2 composed of either an ‘accelerating’ β₃-subunits or a ‘decelerating’ β_2a. These studies revealed a relation between the rate of β-mediated inactivation and PAA-sensitivity (fig.6A-C).

Figure 6

Auxiliary β-subunits modulate inactivation and apparent sensitivity for gallopamil and mibefradil. A) Representative currents through Ca_v1.2 composed of either β₃ (Ca_v1.2(β₃)) or β_2a-subunits (Ca_v1.2(β₃)). Barium (more...)

Jimenez et al³⁰ demonstrated that inhibition of Ca_v2.1, Ca_v2.3 and Ca_v1.2 by MIB is significantly stronger if the corresponding α₁-subunits are coexpressed with an ‘accelerating’ β_1b rather than with a ‘decelerating’ β_2a-subunit. The concentration of drug required to block Ca_v2.1 and Ca_v2.3 composed of β_2a was approximately 10-fold higher (fig. 6D, left, middle). A smaller difference was observed for Ca_v1.2 (fig. 6D, right). Further evidence for a role of the β-subunit interaction in Ca²⁺ channel block comes from Zamponi et al³³ who reported that coexpression of the Ca_v2.3 α₁ subunit with β_2a impaired channel block by piperidines compared to Ca_v2.3 coexpressed with the ‘accelerating’ β_1b. In summary, these data suggest that a β-subunit-mediated increase or reduction in voltage-dependent inactivation modulates Ca_v channel inhibition by PAAs, MIB and piperidines.

Inactivation Determinants and DHP Sensitivity

Evidence for a key role of inactivation in channel inhibition by DHPs came from the drug-induced shift of Ca_v1.2 availability to hyperpolarized voltages in myocardial and smooth muscle cells. This is conventionally explained as high affinity binding of drugs such as nifedipine or isradipine to the inactivated channel conformation.^20,21 Ca_v1.2 composed of ‘accelerating’ β_1a- (Ca_v1.2(β_1a)) or β₃-subunits (Ca_v1.2(β₃)) were, therefore, expected to enhance DHP-binding and channel block.

However, Lacinova et al³¹ reported almost identical sensitivities of Ca_v1.2(β₃) and Ca_v1.2 channels lacking the β subunit for (+)isradipine, despite the considerable differences in inactivation.

Berjukow et al³⁴ came to a similar conclusion by comparing the DHP sensitivities of Ca_v channels containing either β_1a or β_2a subunits (fig.7). A DHP-sensitive Ca_v2.1 mutant composed of the ‘accelerating’ β_1a (α_1A-DHP/β_1a) displayed at —80 mV a 3-fold higher steady-state fraction of channels in an inactivated state compared to slowly inactivating α_1A-DHP/β_2a. This larger fraction of hypothetically high affinity drug receptors did, however, not promote additional channel block by (+)isradipine (fig. 7A). Analogous observations were made for Ca_v1.2, the prime target of DHP in vascular smooth muscle cells (fig. 7).³⁴

Figure 7

The rapidly inactivating Ca_v2.1 mutant α_1A-DHP displays a higher DHP-sensitivity than wild-type Ca_v1.2. A) Concentration-response relationships of peak barium current inhibition of α_1A-DHP(β_1a) (open squares), α_1A-DHP(β (more...)

There is, nevertheless, evidence for an important role of the inactivation machinery in channel block by DHPs. The more rapidly inactivating mutant α_1A-DHP displays a higher sensitivity for (+)isradipine than the slower inactivating wild-type Ca_v1.2, irrespective whether an ‘accelerating’ β_1a or a ‘decelerating’ β_2a-subunit were coexpressed (fig. 7A). It is, therefore, tempting to speculate that the higher DHP-sensitivity of α_1A-DHP is related to their faster inactivation compared to Ca_v1.2 (fig. 7B,C).

Furthermore, Berjukow et al³⁴ demonstrated that substitution of the putative DHP-binding determinant I1471 in segment IVS6 (fig. 1D) by alanine slowed the rate of fast voltage-dependent inactivation (fig. 8A,B) and additionally accelerated recovery of drug modified channels from inactivation. It appears that mutation I1471A destabilises the intrinsic fast inactivated state and the DHP-induced state.

Figure 8

Onset of DHP-induced inactivation is voltage-dependent and recovery is modulated by inactivation determinants. A) (+)Isradipine (1μM) accelerates barium current decay through L-type construct Ca_v1.2(β1a) (traces on the left) and slows (more...)

A detailed study of kinetics of entry into and recovery from inactivation and DHP-inhibition of various Ca_v1.2 and Ca_v2.1 mutants led us to a hypothesis that these drugs promote fast voltage-dependent inactivation without binding selectively to this state. This idea was further supported by (+)-isradipine action on Ca_v1.2 mutant V1477A that is almost completely lacking fast voltage-dependent inactivation. (+)-Isradipine accelerated the current decay in a voltage-dependent manner comparable to voltage dependence of inactivation of wild type Ca_v1.2 (fig. 8C). The effect of (+)-isradipine on this channel can, therefore, be interpreted as a restoration of fast voltage-dependent inactivation.³⁵

On the Role of Ca²⁺-Dependent Inactivation in Drug Sensitivity

Ca²⁺-dependent inactivation causes an additional rapid component in current decay through Ca_v1.2 channels (fig. 1B and 9A). Sokolov et al³² reported that accelerated inactivation with Ca²⁺ as charge carrier (compared to Ba²⁺) is accompanied by enhanced use-dependent block by (-)gallopamil (fig.9B). This effect was apparently caused by a slower recovery from channel block in the presence of Ca²⁺ (fig. 9C). Its molecular basis has yet to be elucidated.

Figure 9

On the role of Ca²⁺ dependent inactivation in (-)gallopapil action. A) Normalised Ca²⁺ currents through Ca_v1.2(β_2a) elicited by a 0.5 Hz train of 10 pulses (-80 mV to 0 mV) in the presence of 10 μM (-)gallopamil. B) Use-dependent peak (more...)

Simulation of the Drug-Channel Interaction

Different versions of the “modulated receptor paradigm”³⁶ explain the cumulative Ca²⁺ channel block by PAA, DIL and MIB during a train of test pulses (“use-dependent block”, fig. 10) by selective drug binding to either open or inactivated channels.^20,21,37

Figure 10

Simulation of use-dependent inhibition of Ca_v1.2 by (-)gallopamil. A, B) Use-dependent Ca²⁺ channel block of Ca_v1.2(β₃) (A, left panel) and Ca_v1.2(β_2a) (B, left panel) by 10μM (-)gallopamil (same experiments as in fig.6B). The (more...)

Such a scenario would imply that during a single pulse the drug “has not enough time” to bind to all channels. Thus, due to the slow drug binding only a small fraction of channels would be blocked. Upon repolarization the channels recover to the low affinity resting state where the drug dissociates from its receptor site (fig. 10). Drug unbinding is, however, slow as well and a certain fraction of channels will remain blocked until the next pulse is applied leading finally to cumulative (use-dependent) channel inhibition.

Alternatively, drug binding could occur independently of the channel state. In such a scenario that we call “drug inactivation synergism,”³⁸ Ca²⁺ antagonists accelerate channel transitions into the fast inactivated state like ‘accelerating’ β-subunits or point mutations.

Figure 10 illustrates the two competing models. Both of them satisfactory reproduce the experimental data. In the “drug inactivation synergism” model the effect of (-)gallopamil on Ca_v1.2(β₃) and Ca_v1.2(β_2a) is simulated by an about 7-fold increase in the on-rate of fast voltage-dependent inactivation (rate α(PAA)) and an 8-fold decrease in recovery from drug-induced inactivation (rate β(PAA)). The difference in use-dependent inhibition of the two β-subunit compositions arise, therefore, solely due to the differences in intrinsic inactivation in Ca_v1.2(β₃) and Ca_v1.2(β_2a), i.e., the “drug-inactivation synergism” model reproduces the differences in apparent drug sensitivity without the necessity to assume different drug affinities for Ca_v1.2(β₃) and Ca_v1.2(β_2a).

In the “open channel block” model we had to presume a higher affinity of (-)gallopamil for rapidly inactivating Ca_v1.2(β₃) compared to Ca_v1.2(β_2a) (k_on[D](Ca_v(β₃)) = 0.5 s^-1 vs. k_on[D](Ca_v(β_2a)) = 0.15 s^-1). This model reproduces the data only if we postulate a direct effect of the β-subunit on the drug binding process. Moreover, if we hypothesise equal affinities this model predicts enhanced inhibition of slowly inactivating (open) Ca_v channels and not—as observed in most experiments—stronger block of faster inactivating channels (figs. 2-7).

Summary and Outlook

Point mutations in different parts of the Ca_v α₁-subunits that either enhance or impair channel inactivation simultaneously increase or reduce channel inhibition by PAA, DIL, MIB and to a certain extent by DHPs. Fast inactivating channels tend to be more efficiently inhibited than slow-inactivating ones. This simple correlation has been confirmed by independent groups for wild type, chimeric or mutant HVA Ca_v.^25-33 A similar conclusion can be drawn the drug action on Ca_v channels containing either ‘accelerating’ (e.g., β₁ or β₃) or ‘decelerating’ (β_2a) β-subunits.^30,32 Subunit compositions promoting faster channel inactivation increase the apparent drug sensitivity.

A possible scenario of a “drug inactivation synergism” is illustrated in (fig. 10) This model does not exclude state-dependent drug access for partially charged compounds. However, once these drugs dock within their binding pockets they are more likely to promote inactivation than to plug the open channel pore (fig.10). Some amino acids (e.g., I1470, I1471 in segment IVS6 and the IFV motif close to the inner channel mouth in segment IIIS6 of Ca_v1.2 as well as M1464 and A1467 in Ca_v2.1 environment (fig. 1D)) are strong inactivation determinants.

It is currently not entirely clear which of the determinants of drug sensitivity in segments IIIS5, IIIS6 and IVS6 form part of the drug binding pocket and which of them affect the drug-channel interaction in an indirect manner via inactivation (fig. 1D). In functional studies the effects of mutations on PAA-, DIL- and MIB-sensitivity is indistinguishable from mutations localised outside the putative drug-binding region. The respective impact of IIIS5, IIIS6 and IVS6 amino acids in inactivation and drug binding has yet to be thoroughly elucidated.

Acknowledgements

We Thank Jörg Mitterdorfer for Comments on the Manuscript. Supported by FWF grant 15914.

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