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 voltagedependent). 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 À140 mV. Conclusion and implications: Drug molecules dissociate at different rates from open and closed HERG channels ('usedependent' 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.

Light stimuli produce graded hyperpolarizations of the photoreceptor plasma membrane and an associated decrease in a voltagegated calcium channel conductance that mediates release of glutamate neurotransmitter. The Ca v1.4 channel is thought to be involved in this process. The CACNA1F gene encodes the poreforming subunit of the Cav1.4 channel and various mutations in CACNA1F cause X-linked incomplete congenital stationary night blindness (CSNB2). The molecular mechanism of the pathology underlying the CSNB2 phenotype remains to be established. Recent clinical investigations of a New Zealand family found a severe visual disorder that has some clinical similarities to, but is clearly distinct from, CSNB2. Here, we report investigations into the molecular mechanism of the pathology of this condition. Molecular genetic analyses identified a previously undescribed nucleotide substitution in CACNA1F that is predicted to encode an isoleucine to threonine substitution at CACNA1F residue 745. The I745T CACNA1F allele produced a remarkable approximately ؊30-mV shift in the voltage dependence of Cav1.4 channel activation and significantly slower inactivation kinetics in an expression system. These findings imply that substitution of this wild-type residue in transmembrane segment IIS6 may have decreased the energy required to open the channel. Collectively, these findings suggest that a gain-of-function mechanism involving increased Cav1.4 channel activity is likely to cause the unusual phenotype.

The entry of Ca 2ϩ through voltage-gated Ca 2ϩ channels has direct effects on muscle contraction, release of hormones and neurotransmitters, hearing, vision, gene expression, and other important physiological functions (2). The pore-forming ␣ 1 -subunits of voltage-gated Ca 2ϩ channels are composed of four homologous domains formed by six transmembrane segments (S1-S6) that are linked together on a single polypeptide (3). A membrane depolarization initiates channel openings (activation) and closures (inactivation). These events can be considered a multistep process consisting of a conformational change in the voltage sensor, a transmission of the signal to the pore region, the opening of the pore, and channel closure due to inactivation. The voltage-sensing machinery is formed by multiple charged amino acids located in segment S4 and adjacent structures of each domain (4). A large number of amino acids involved in Ca 2ϩ channel inactivation have been identified and several molecular mechanisms for this process have been proposed (for reviews see Refs. 5-7).The molecular mechanism of the voltage-dependent pore opening of Ca 2ϩ channels, however, is less studied and largely unknown. The first attempt to localize the structural elements in Ca 2ϩ channel ␣ 1 -subunits that are involved in channel activation was made by Tanabe et al. (8) who constructed chimeric channels in which sequence stretches of a slow activating ("skeletal muscle-like") Ca V 1.1 ␣ 1 -subunit were replaced by sequences from a fast activating ("cardiac-like") Ca V 1.2 ␣ 1 -subunit. The chimeras activated slowly if repeat I of the Ca V 1.2 ␣ 1 -subunit was replaced by the Ca V 1.1 ␣ 1 -sequence. In a later study, replacement of domains I, II, and III of the low voltage and fast activating Ca V 3.1 ␣ 1 -subunit with the corresponding domains of the high voltage-activated Ca V 1.2 ␣ 1 -subunit resulted in a high voltage-activated channel (9). An important role of domains I and III but not II and IV on midpoint voltage and time constants of activation was reported by Garcia et al. (10) who mutated the arginines in the S4 segments of all four domains of a chimeric channel to neutral or negative amino acids. The removal of prolines that are conserved in segments IS4 and IIIS4 of voltage-gated Ca 2ϩ channels resulted in shortening of channel open time, whereas introduction of extra prolines to corresponding positions of IIS4 and IVS4 lengthened the channel open time (11).Our present study was initiated by the recent finding that a novel retinal disorder is caused by a point mutation (I745T) in segment IIS6 of the Ca V 1.4 ␣ 1 -subunit that shifts the voltage dependence of Ca V 1.4 channel activation by approximately Ϫ30 mV (1, 12). As Ca V 1.4 channels express only at low density in mammalian cell lines (13) we have decided to study the functional roles of this residue and neighboring residues in segment IIS6 by introducing and characterizing mutations in the homologous Ca V 1.2 channel. Our findings demonstrate that residue Ile-781 and three neigh...

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 (Cav2.1) 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 α1‐subunits and by interactions with other channel subunits. This review focuses on structural determinants of Ca2+ channel inactivation in different parts of Ca2+ channel α1‐subunits, including pore‐forming transmembrane segments and loops, intracellular domain linkers and the carboxyl terminus. Inactivation is also affected by the interaction of the α1‐subunits with auxiliary β‐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.

To investigate the molecular basis of the calcium channel block by diltiazem, we transferred amino acids of the highly sensitive and stereoselective L-type (␣ 1S or ␣ 1C ) to a weakly sensitive, nonstereoselective class A (␣ 1A ) calcium channel. Transfer of three amino acids of transmembrane segment IVS6 of L-type ␣ 1 into the ␣ 1A 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.L-type calcium (Ca 2ϩ ) channels (classes C (formed by ␣ 1C subunits), D (␣ 1D ), and S (␣ 1S )) possess high affinity stereoselective drug receptors for Ca 2ϩ antagonists such as 1,4-dihydropyridines (DHPs), 1 phenylalkylamines (PAAs), and benzothiazepines (BTZs) (reviewed in Refs. 1-5) located on their pore-forming ␣ 1 channel subunit (6). Classes A (␣ 1A ), B (␣ 1B ), and E (␣ 1E ) Ca 2ϩ channels are insensitive for DHPs (2, 5, 7-9) and only weakly sensitive for . Essential parts of the high affinity binding sites for DHPs and PAAs on L-type Ca 2ϩ channels have been identified by replacing sequence stretches in ␣ 1C or ␣ 1S subunits by corresponding non-L-type sequences (8, 13) or by mutating single amino acids in these subunits (10, 13). Alternatively, molecular determinants of the high affinity DHP and PAA receptor sites could be localized in pore-lining regions of repeats III and/or IV by transferring L-type ␣ 1 sequences into the ␣ 1A subunit (9, 12). Transfer of segment IVS6 from ␣ 1S to ␣ 1A enhanced PAA sensitivity of the resulting ␣ 1A /␣ 1S chimera to the level of L-type ␣ 1 subunits (12).The efficacy of the BTZ diltiazem as an antiarrhythmic and antihypertensive drug is due to its voltage-and use-dependent block of L-type Ca 2ϩ channels (14). Studies on cloned ␣ 1 subunits of different Ca 2ϩ channel classes (C, B, A, and E) have enabled a more precise characterization of their pharmacological features (15). To identify the molecular determinants of the high affinity BTZ interaction domain of L-type Ca 2ϩ channels, we introduced corresponding L-type sequence stretches into ␣ 1A . The diltiazem sensitivity of the resulting ␣ 1 chimeras was measured as use-dependent barium current (I Ba ) block after coexpression with ␤ 1a (16) and ␣ 2 /␦ (17) in Xenopus oocytes. EXPERIMENTAL PROCEDURESMolecular Biology-The construction of L-type chimera Lh (repeats I-IV from ␣ 1C-a (18)) with the N terminus replaced with ␣ 1S (19), as well as construction of chimeras AL12h and AL22, were described previously (9, 12). Chimera AL20 was generated by replacing the ClaI (nucleotide position, 4925)-XbaI (3Ј-polylinker) fragment of AL9 (9) by ...

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 ISrHEK by the dihydropyridine isradipine was not stereoselective; 1 gM (+) and (-isradipine inhibited the current by 30+4% (n=3) and 29+2% (n=4) respectively. (+)-Isradipine and (-)-isradipine (10 giM) inhibited ISr-HEK by 89 + 4% (n =5) and 88 + 8% (n = 3) respectively. The 7-bromo substituted (±)-isradipine (V02605, 10 giM) which is almost inactive on L-type calcium channels also inhibited ISr-HEK (83+9%, n=3) as was observed for 10 kIM (-)-nimodipine (78+6%, n=5). Interestingly, 10 gIM (±)-Bay K 8644 (n =5) had no effect on the current. ISr-HEK was only slightly inhibited by the cone snail toxins wo-CTx GVIA (1 gM, inhibition by 17 + 3%, n = 4) and Cw-CTx MVIIC(1 gM, inhibition by 20 + 3%, n = 4). The funnel web spider toxin w-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 oa, 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.

The role of channel inactivation in the molecular mechanism of calcium (Ca channel block by PAA is, however, still not understood. High affinity determinants of the PAA-receptor site of L-type Ca 2ϩ channels have recently been identified on transmembrane segment IVS6 by mutating putative pore-orientated amino acids and subsequent screening of the resulting mutants for PAA-sensitivity (8) or by transferring the responsible L-type amino acids into the ␣ 1A subunit of P͞Q-type channels thereby introducing PAA sensitivity (10, 11). The resulting highly PAA sensitive triple ␣ 1A mutant AL25 (11) displayed significantly faster inactivation kinetics than the wild-type channel. Furthermore, an even more rapid inactivation and higher apparent PAA sensitivity was induced into ␣ 1A if an additional phenylalanine in position 1,805 of AL25 was mutated to the corresponding L-type methionine in IVS6 suggesting a close link between inactivation properties of the channel and use-dependent block by PAA.However, because mutations of the PAA-receptor site itself or of closely located amino acids may not only affect channel inactivation but also the interaction of PAAs with their binding site on transmembrane segment IVS6, no unequivocal conclusions about the role of inactivation in channel block could be drawn (11). To answer this question we designed Ca 2ϩ channels that inactivate at different rates by site directed mutagenesis of segment IIIS6.We report here that substitutions of putative pore-lining amino acids that are highly conserved between ␣ 1A , ␣ 1S , and ␣ 1C gradually reduce the rate of channel inactivation while also gradually reducing use-dependent block by (Ϫ)D600. Our data indicate that changes in Ca 2ϩ channel inactivation induced by site directed mutagenesis modulate use-dependent Ca 2ϩ channel block by PAA. This suggests that channel inactivation is an important determinant of use-dependent Ca 2ϩ channel block by these drugs.

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