State dependent dissociation of HERG channel inhibitors.

BACKGROUND AND PURPOSE: Inhibition of HERG channels prolongs the ventricular action potential and the QT interval with the risk of torsade de pointes arrhythmias and sudden cardiac death. Many drugs induce greater inhibition of HERG channels when the cell membrane is depolarized frequently. The dependence of inhibition on the pulsing rate may yield different IC(50) values at different frequencies and thus affect the quantification of HERG channel block. We systematically compared the kinetics of HERG channel inhibition and recovery from block by 8 blockers at different frequencies. EXPERIMENTAL APPROACH: HERG channels were expressed heterologously in Xenopus oocytes and currents were measured with the two-electrode voltage clamp technique. KEY RESULTS: Frequency-dependent block was observed for amiodarone, cisapride, droperidol and haloperidol (group 1) whereas bepridil, domperidone, E-4031 and terfenadine (group 2) induced similar pulse-dependent block at all frequencies. With the group 1 compounds, HERG channels recovered from block in the presence of drug (recovery being voltage-dependent). No substantial recovery from block was observed with the second group of compounds. Washing out of bepridil, domperidone, E-4031 and terfenadine was substantially augmented by frequent pulsing. Mutation D540K in the HERG channel (which exhibits reopening at negative voltages) facilitated recovery from block by these compounds at -140 mV. CONCLUSION AND IMPLICATIONS: Drug molecules dissociate at different rates from open and closed HERG channels ('use-dependent' dissociation). Our data suggest that apparently 'trapped' drugs (group 2) dissociated from the open channel state whereas group 1 compounds dissociated from open and resting states.

On the fate of skeletal myoblasts in a cardiac environment: down-regulation of voltage-gated ion channels.

We have analysed the voltage-gated ion channels and fusion competence of skeletal muscle myoblasts labelled with green fluorescent protein (GFP) and the membrane dye PKH transplanted into the infarcted myocardium of syngenic rats. After cell transplantation the animals were killed and GFP(+)-PKH(+) myoblasts enzymatically isolated for subsequent studies of ionic currents through voltage-gated sodium, calcium and potassium channels. A down-regulation of all three types of ion channels after engraftment was observed. The fraction of cells with calcium (68%) and sodium channels (65%) declined to zero within 24 h and 1 week, respectively. Down-regulation of potassium currents (90% in control) occurred within 2 weeks to about 30%. Before injection myoblasts expressed predominantly transient outward potassium channels whereas after isolation from the myocardium exclusively rapid delayed rectifier channels. The currents recovered completely between 1 and 6 weeks under cell culture conditions. The down-regulation of ion channels and changes in potassium current kinetics suggest that the environment provided by infarcted myocardium affects expression of voltage-gated ion channels of skeletal myoblasts.

Inactivation determinants in segment IIIS6 of Ca(v)3.1.

1. Low threshold, T-type, Ca(2+) channels of the Ca(v)3 family display the fastest inactivation kinetics among all voltage-gated Ca(2+) channels. The molecular inactivation determinants of this channel family are largely unknown. Here we investigate whether segment IIIS6 plays a role in Ca(v)3.1 inactivation as observed previously in high voltage-activated Ca(2+) channels. 2. Amino acids that are identical in IIIS6 segments of all Ca(2+) channel subtypes were mutated to alanine (F1505A, F1506A, N1509A, F1511A, V1512A, F1519A, FV1511/1512AA). Additionally M1510 was mutated to isoleucine and alanine. 3. The kinetic properties of the mutants were analysed with the two-microelectrode voltage-clamp technique after expression in Xenopus oocytes. The time constant for the barium current (I(Ba)) inactivation, tau(inact), of wild-type channels at -20 mV was 9.5 +/- 0.4 ms; the corresponding time constants of the mutants ranged from 9.2 +/- 0.4 ms in V1512A to 45.7 +/- 5.2 ms (4.8-fold slowing) in M1510I. Recovery at -80 mV was most significantly slowed by V1512A and accelerated by F1511A. 4. We conclude that amino acids M1510, F1511 and V1512 corresponding to previously identified inactivation determinants in IIIS6 of Ca(v)2.1 (Hering et al. 1998) have a significant role in Ca(v)3.1 inactivation. These data suggest common elements in the molecular architecture of the inactivation mechanism in high and low threshold Ca(2+) channels.

Voltage-dependent acceleration of Ca(v)1.2 channel current decay by (+)- and (-)-isradipine.

Inhibition of Ca(v)1.2 by antagonist 1,4 dihydropyridines (DHPs) is associated with a drug-induced acceleration of the calcium (Ca(2+)) channel current decay. This feature is contradictorily interpreted as open channel block or as drug-induced inactivation. To elucidate the underlying molecular mechanism we investigated the effects of (+)- and (-)-isradipine on Ca(v)1.2 inactivation gating at different membrane potentials. alpha(1)1.2 Constructs were expressed together with alpha(2)-delta- and beta(1a)- subunits in Xenopus oocytes and drug-induced changes in barium current (I(Ba)) kinetics analysed with the two microelectrode voltage clamp technique. To study isradipine effects on I(Ba) decay without contamination by intrinsic inactivation we expressed a mutant (V1504A) lacking fast voltage-dependent inactivation. At a subthreshold potential of -30 mV a 200-times higher concentration of (-)-isradipine was required to induce a comparable amount of inactivation as by (+)-isradipine. At +20 mV the two enantiomers were equally efficient in accelerating the I(Ba) decay. Faster recovery from (-)- than from (+)-isradipine-induced inactivation at -80 mV in a Ca(v)1.2 construct (tau((-)-isr.(Cav1.2))=0.74 s

Amino acids in segment IVS6 and beta-subunit interaction support distinct conformational changes during Ca(v)2.1 inactivation.

Ca(v)2.1 mediates voltage-gated Ca2+ entry into neurons and the release of neurotransmitters at synapses of the central nervous system. An inactivation process that is modulated by the auxiliary beta-subunits regulates Ca2+ entry through Ca(v)2.1. However, the molecular mechanism of this alpha1-beta-subunit interaction remains unknown. Herein we report the identification of new determinants within segment IVS6 of the alpha(1)2.1-subunit that markedly influence channel inactivation. Systematic substitution of residues within IVS6 with amino acids of different size, charge, and polarity resulted in mutant channels with rates of fast inactivation (k(inact)) ranging from a 1.5-fold slowing in V1818I (k(inact) = 0.98 +/- 0.09 s(-1) compared with wild type alpha(1)2.1/alpha2-delta/beta1a k(inact) = 1.35 +/- 0.25 s(-1) to a 75-fold acceleration in mutant M1811Q (k(inact) = 102 +/- 3 s(-1). Coexpression of mutant alpha(1)2.1-subunits with beta(2a) resulted in two different phenotypes of current inactivation: 1) a pronounced reduction in the rate of channel inactivation or 2) an attenuation of a slow component in I(Ba) inactivation. Simulations revealed that these two distinct inactivation phenotypes arise from a beta2a-subunit-induced destabilization of the fast-inactivated state. The IVS6- and beta2a-subunit-mediated effects on Ca(v)2.1 inactivation are likely to occur via independent mechanisms.

Molecular determinants of inactivation in voltage-gated Ca2+ channels.

Evolution has created a large family of different classes of voltage-gated Ca2+ channels and a variety of additional splice variants with different inactivation properties. Inactivation controls the amount of Ca2+ entry during an action potential and is, therefore, believed to play an important role in tissue-specific Ca2+ signalling. Furthermore, mutations in a neuronal Ca2+ channel (Ca(v)2.1) that are associated with the aetiology of neurological disorders such as familial hemiplegic migraine and ataxia cause significant changes in the process of channel inactivation. Ca2+ channels of a given subtype may inactivate by three different conformational changes: a fast and a slow voltage-dependent inactivation process and in some channel types by an additional Ca2+-dependent inactivation mechanism. Inactivation kinetics of Ca2+ channels are determined by the intrinsic properties of their pore-forming alpha1-subunits and by interactions with other channel subunits. This review focuses on structural determinants of Ca2+ channel inactivation in different parts of Ca2+ channel alpha1-subunits, including pore-forming transmembrane segments and loops, intracellular domain linkers and the carboxyl terminus. Inactivation is also affected by the interaction of the alpha1-subunits with auxiliary beta-subunits and intracellular regulator proteins. The evidence shows that pore-forming S6 segments and conformational changes in extra- (pore loop) and intracellular linkers connected to pore-forming segments may play a principal role in the modulation of Ca2+ channel inactivation. Structural concepts of Ca2+ channel inactivation are discussed.

Molecular mechanism of calcium channel block by isradipine. Role of a drug-induced inactivated channel conformation.

The role of the inactivated channel conformation in the molecular mechanism of Ca(2+) channel block by the 1,4-dihydropyridine (DHP) (+)-isradipine was analyzed in L-type channel constructs (alpha(1Lc); Berjukow, S., Gapp, F., Aczel, S., Sinnegger, M. J., Mitterdorfer, J., Glossmann, H., and Hering, S. (1999) J. Biol. Chem. 274, 6154-6160) and a DHP-sensitive class A Ca(2+) channel mutant (alpha(1A-DHP); Sinnegger, M. J., Wang, Z., Grabner, M., Hering, S., Striessnig, J., Glossmann, H., and Mitterdorfer, J. (1997) J. Biol. Chem. 272, 27686-27693) carrying the high affinity determinants of the DHP receptor site but inactivating at different rates. Ca(2+) channel inactivation was modulated by coexpressing the alpha(1A-DHP)- or alpha(1Lc)-subunits in Xenopus oocytes with either the beta(2a)- or the beta(1a)-subunit and amino acid substitutions in L-type segment IVS6 (I1497A, I1498A, and V1504A). Contrary to a modulated receptor mechanism assuming high affinity DHP binding to the inactivated state we observed no clear correlation between steady state inactivation and Ca(2+) channel block by (+)-isradipine: (i) a 3-fold larger fraction of alpha(1A-DHP)/beta(1a) channels in steady state inactivation at -80 mV (compared with alpha(1A-DHP)/beta(2a)) did not enhance the block by (+)-isradipine; (ii) different steady state inactivation of alpha(1Lc) mutants at -30 mV did not correlate with voltage-dependent channel block; and (iii) the midpoint-voltages of the inactivation curves of slowly inactivating L-type constructs and more rapidly inactivating alpha(1Lc)/beta(1a) channels were shifted to a comparable extent to more hyperpolarized voltages. A kinetic analysis of (+)-isradipine interaction with different L-type channel constructs revealed a drug-induced inactivated state. Entry and recovery from drug-induced inactivation are modulated by intrinsic inactivation determinants, suggesting a synergism between intrinsic inactivation and DHP block.

Sequence differences between alpha1C and alpha1S Ca2+ channel subunits reveal structural determinants of a guarded and modulated benzothiazepine receptor.

The molecular basis of the Ca2+ channel block by (+)-cis-diltiazem was studied in class A/L-type chimeras and mutant alpha1C-a Ca2+ channels. Chimeras consisted of either rabbit heart (alpha1C-a) or carp skeletal muscle (alpha1S) sequence in transmembrane segments IIIS6, IVS6, and adjacent S5-S6 linkers. Only chimeras containing sequences from alpha1C-a were efficiently blocked by (+)-cis-diltiazem, whereas the phenylalkylamine (-)-gallopamil efficiently blocked both constructs. Carp skeletal muscle and rabbit heart Ca2+ channel alpha1 subunits differ with respect to two nonconserved amino acids in segments IVS6. Transfer of a single leucine (Leu1383, located at the extracellular mouth of the pore) from IVS6 alpha1C-a to IVS6 of alpha1S significantly increased the (+)-cis-diltiazem sensitivity of the corresponding mutant L1383I. An analysis of the role of the two heterologous amino acids in a L-type alpha1 subunit revealed that corresponding amino acids in position 1487 (outer channel mouth) determine recovery of resting Ca2+ channels from block by (+)-cis-diltiazem. The second heterologous amino acid in position 1504 of segment IVS6 (inner channel mouth) was identified as crucial inactivation determinant of L-type Ca2+ channels. This residue simultaneously modulates drug binding during membrane depolarization. Our study provides the first evidence for a guarded and modulated benzothiazepine receptor on L-type channels.

Molecular mechanism of use-dependent calcium channel block by phenylalkylamines: role of inactivation.

The role of channel inactivation in the molecular mechanism of calcium (Ca2+) channel block by phenylalkylamines (PAA) was analyzed by designing mutant Ca2+ channels that carry the high affinity determinants of the PAA receptor site [Hockerman, G. H., Johnson, B. D., Scheuer, T., and Catterall, W. A. (1995) J. Biol. Chem. 270, 22119-22122] but inactivate at different rates. Use-dependent block by PAAs was studied after expressing the mutant Ca2+ channels in Xenopus oocytes. Substitution of single putative pore-orientated amino acids in segment IIIS6 by alanine (F-1499-A, F-1500-A, F-1510-A, I-1514-A, and F-1515-A) gradually slowed channel inactivation and simultaneously reduced inhibition of barium currents (I(Ba)) by (-)D600 upon depolarization by 100 ms steps at 0.1 Hz. This apparent reduction in drug sensitivity was only evident if test pulses were applied at a low frequency of 0.1 Hz and almost disappeared at the frequency of 1 Hz. (-)D600 slowed I(Ba) recovery after maintained membrane depolarization (1-3 sec) to a comparable extent in all channel constructs. A drug-induced delay in the onset of I(Ba) recovery from inactivation suggests that PAAs promote the transition to a deep inactivated channel conformation. These findings indicate that apparent PAA sensitivity of Ca2+ channels is not only defined by drug interaction with its receptor site but also crucially dependent on intrinsic gating properties of the channel molecule. A molecular model for PAA-Ca2+ channel interaction that accounts for the relationship between drug induced inactivation and channel block by PAA is proposed.

Calcium channel block by (-)devapamil is affected by the sequence environment and composition of the phenylalkylamine receptor site.

The pore-forming alpha 1 subunit of L-type calcium (Ca2+) channels is the molecular target of Ca2+ channel blockers such as phenylalkylamines (PAAs). Association and dissociation rates of (-)devapamil were compared for a highly PAA-sensitive L-type Ca2+ channel chimera (Lh) and various class A Ca2+ channel mutants. These mutants carry the high-affinity determinants of the PAA receptor site in a class A sequence environment. Apparent drug association and dissociation rate constants were significantly affected by the sequence environment (class A or L-type) of the PAA receptor site. Single point mutations affecting the high-affinity determinants in segments IVS6 of the PAA receptor site, introduced into a class A environment, reduced the apparent drug association rates. Mutation I1811M in transmembrane segment IVS6 (mutant AL25/-I) had the highest impact and decreased the apparent association rate for (-)devapamil by approximately 30-fold, suggesting that this pore-lining isoleucine in transmembrane segment IVS6 plays a key role in the formation of the PAA receptor site. In contrast, apparent drug dissociation rates of Ca2+ channels in the resting state were almost unaffected by point mutations of the PAA receptor site.

Transfer of high sensitivity for benzothiazepines from L-type to class A (BI) calcium channels.

To investigate the molecular basis of the calcium channel block by diltiazem, we transferred amino acids of the highly sensitive and stereoselective L-type (alpha1S or alpha1C) to a weakly sensitive, nonstereoselective class A (alpha1A) calcium channel. Transfer of three amino acids of transmembrane segment IVS6 of L-type alpha1 into the alpha1A subunit (I1804Y, S1808A, and M1811I) was sufficient to support a use-dependent block by diltiazem and by the phenylalkylamine (-)-gallopamil after expression in Xenopus oocytes. An additional mutation F1805M increased the sensitivity for (-)-gallopamil but not for diltiazem. Our data suggest that the receptor domains for diltiazem and gallopamil have common but not identical molecular determinants in transmembrane segment IVS6. These mutations also identified single amino acid residues in segment IVS6 that are important for class A channel inactivation.

Endogenous calcium channels in human embryonic kidney (HEK293) cells.

1. We have identified endogenous calcium channel currents in HEK293 cells. Whole cell endogenous currents (ISr-HEK) were studied in single HEK293 cells with 10 mM strontium as the charge carrier by the patch clamp technique. The kinetic properties and pharmacological features of ISr-HEK were characterized and compared with the properties of a heterologously expressed chimeric L-type calcium channel construct. 2. ISr-HEK activated on depolarization to voltages positive of -40 mV. It had transient current kinetics with a time to peak of 16 +/- 1.4 ms (n = 7) and an inactivation times constant of 52 +/- 5 ms (n = 7) at a test potential of 0 mV. The voltage for half maximal activation was -19.0 +/- 1.5 mV (n = 7) and the voltage for half maximal steady-state inactivation was -39.7 +/- 2.3 mV (n = 7). 3. Block of ISr-HEK by the dihydropyridine isradipine was not stereoselective; 1 microM (+) and (-)-isradipine inhibited the current by 30 +/- 4% (n = 3) and 29 +/- 2% (n = 4) respectively. (+)-Isradipine and (-)-isradipine (10 microM) inhibited ISr-HEK by 89 +/- 4% (n = 5) and 88 +/- 8% (n = 3) respectively. The 7-bromo substituted (+/-)-isradipine (VO2605, 10 microM) which is almost inactive on L-type calcium channels also inhibited ISr-HEK (83 +/- 9%, n = 3) as was observed for 10 microM (-)-nimodipine (78 +/- 6%, n = 5). Interestingly, 10 microM (+/-)-Bay K 8644 (n = 5) had no effect on the current. ISr-HEK was only slightly inhibited by the cone snail toxins omega-CTx GVIA (1 microM, inhibition by 17 +/- 3%, n = 4) and omega-CTx MVIIC (1 microM, inhibition by 20 +/- 3%, n = 4). The funnel web spider toxin omega-Aga IVA (200 nM) inhibited ISr-HEK by 19 +/- 2%, n = 4). 4. In cells expressing ISr-HEK, maximum inward current densities of 0.24 +/- 0.03 pA/pF and 0.39 +/- 0.7 pA/ pF (at a test potential of -10 mV) were estimated in two different batches of HEK293 cells. The current density increased to 0.88 +/- 0.18 pA/pF or 1.11 +/- 0.2 pA/pF respectively, if the cells were cultured for 4 days in serum-free medium. 5. Co-expression of a chimeric L-type calcium channel construct revealed that ISr-HEK and L-type calcium channel currents could be distinguished by their different voltage-dependencies and current kinetics. The current density after heterologous expression of the L-type alpha 1 subunit chimera was estimated to be about ten times higher in serum containing medium (2.14 +/- 0.45 pA/pF) than that of ISr-HEK under the same conditions.

Chimeric L-type Ca2+ channels expressed in Xenopus laevis oocytes reveal role of repeats III and IV in activation gating.

1. Chimeric alpha 1 subunits consisting of repeat I and II from the rabbit cardiac (alpha 1C-a) and repeat III and IV from the carp skeletal muscle Ca2+ channel (alpha 1S) were constructed and expressed in Xenopus laevis oocytes without co-expressing other channel subunits. Ba2+-current kinetics of five chimeric channel constructs were studied in Xenopus oocytes using the two-microelectrode technique. 2. Exchange of repeats III and IV of alpha 1C-a with sequences of alpha 1S results in a significantly slower and biexponential activation (apparent activation time constants tau 1act = 19.8 +/- 1.8 ms and tau 2act = 214 +/- 28.7 ms, n = 7) of expressed Ca2+ channel currents; no current inactivation was observable during an 800 ms test pulse to 0 mV. 3. Activation of a chimera consisting of repeats I, II and IV from the alpha 1C-a subunit and repeat III from alpha 1S was fast and monoexponential (tau 1act = 6.33 +/- 1.7 ms, n = 5) and the current inactivated during a 350 ms test pulse to 0 mV (tau inact = 175 +/- 22 ms, n = 5). The current kinetics of this construct did not significantly differ from kinetics of a construct consisting of repeats I to IV from alpha 1C-a (tau 1act = 6.6 +/- 2.1 ms; tau inact = 198 +/- 14 ms; n = 9).(ABSTRACT TRUNCATED AT 250 WORDS)