Calmodulin- And Ca～(2+)-dependent Activity Of The Cav1.2 Ca～(2+) Channels In Guinea-pig Ventricular Myocytes

Calmodulin-and Ca²⁺-dependent activity of the Cav1.2 Ca2+²⁺ channels in guinea-pig ventricular myocytesIntroductionVoltage-gated L-type Ca²⁺ channels mediate the entry of Ca²⁺ into cells in response to membrane depolarization, allowing the activation of numerous Ca²⁺-dependent intracellular events, such as impulse generation, muscle contraction, secretion of hormones and neurotransmitters and gene expression. The activity of many types of calcium channels decreases rapidly when contact between membrane and cytosol is disrupted. This phenomenon, called rundown, is widely spread among voltage-gated L-type Ca²⁺ channels and appears to be related to regulatory factors present in the cytosol, which are rapidly lost upon patch excision. Our recent study indicated that calmodulin (CAM) and ATP are crucial regulatory factors which can reverse rundown of L-type Ca²⁺ channels.CAM is a ubiquitous and major Ca²⁺-binding protein, which directly or indirectly regulates excitation-contraction coupling and other important physiological functions in cardiac myocytes. Cardiac L-type Ca²⁺ channel (Cav1.2) are modulated by the interaction of the channelα₁ subunit C-terminal tail with CaM, such that CAM binding to this region is required for both Ca²⁺-dependent inactivation (CDI) and Ca²⁺-dependent facilitation (CDF) of cardiac L-type Ca²⁺ channel.Recent experiments have implicated direct binding of CaM to L-type channels as a key step in both inactivation and facilitation. CaM exists in cell in two forms, apoCaM (calcium-free CaM) and Ca²⁺/CaM (including Ca²⁺-unsaturated and Ca²⁺-saturated CAM). It has been suggested that apoCaM is preassociated with L-type Ca²⁺ channel and functions as a Ca²⁺ sensor for CDI and CDF of the channel. The dual role of CaM in regulating CDI and CDF suggests that there are multiple binding sites for CaM in the C-terminal tail of L-type Ca²⁺ channel. Several sequences have been implicated as contributing to Ca²⁺/CaM binding to this region of the pore-forming subunit. A sequence designated the IQ motif is demonstrated to be required for both CDI and CDF. In additional, several other sequences, such as A motif, C motif, EF hand and N terminus, have also been implicated in CaM binding, demonstrating that the molecular mechanism of CaM-mediated regulation of Ca²⁺ channel is complicated.In this study, we have examined the calcium- and dose-dependent effects of CaM on L-type Ca²⁺ channels in inside-out patch mode in order to further elucidate the molecular mechanism of CaM-mediated regulation.Methods1. Preparation of single cardiac myocytesA guinea-pig (weight 300-500 g) was anaesthetized with pentobarbital sodium (30 mg/kg ip), and the aorta was cannulated in situ under artificial respiration. Single ventricular myocytes from guinea-pig hearts were dispersed by collagenase. The isolated cells were stored at 4℃in storage solution until used for the patch-clamp experiments.2. Preparation of CaM and CaM₁₂₃₄The coding sequences of CaM were generated by RT-PCR amplification from HEK 293 cells and cloned into pGEX6P-3 (GE Healthcare, Milwaukee, WI, USA). The Ca²⁺-insensitive CaM mutant CaM₁₂₃₄ was created by site-directed mutations of consensus EF-hand motifs, E31A, E67A, S101F, E140A, using QuickChange (Stratagene, La Jolla, CA, USA) from the wild-type construct. CaM and CaM₁₂₃₄ were expressed as glutathione S-transferase (GST) fusion proteins in E. coli BL21 (DE3) and purified using Glutathione Sepharose 4B (GE Healthcare). The GST regions of CaM and CaM₁₂₃₄ were removed by cleavage using PreScission Protease (GE Healthcare). Finally, expressed peptides were checked on SDS-PAGE and quantified by the Bradford method.3. Patch clamp and data analysisCa²⁺ channel activity was monitored with the patch-clamp technique. First, the cell-attached mode was formed and Ca²⁺ channel activity was recorded for 2 min, then the membrane patch was excised from the cell to establish the inside-out patch configuration. One minute after the patch excision, test solutions were applied by moving the patch into a small inset in the perfusion chamber and recorded for 20 min. Barium currents through the Ca²⁺ channel were elicited by depolarizing pulses from -70 to 0 mV for 200 ms duration at a rate of 0.5 Hz. They were recorded with a patch-clamp amplifier (Axopatch-200B, Axon Instruments, USA), and fed to a computer at a sampling rate of 3.3 kHz. The capacity and leakage currents in the current traces were digitally subtracted. The mean current during the period 5-105 ms after the onset of the test pulses (I) was measured and divided by the unitary current amplitude (i) to yield NPo (since I = N×Po×i), where N is the number of channels in the patch and Po is the time-averaged open-state probability of the channels.Data are presented as mean±SE. Student's t-test was used to estimate statistical significance and values of P＜0.05 were considered significant.Results1. Effect of CaM + ATP on Ca²⁺ channel activity in inside-out patch mode.We first examined the effect of CaM on Ca²⁺ channel activity in inside-out patch mode in which Ca²⁺ channels usually showed rundown. At free [Ca²⁺] of 100 nM, after application of CaM + ATP within 1 min after patch excision, Ca²⁺ channel activity was evoked once again. Low and moderate concentration of CaM (0.7, 1.4, 2.1μM) induced channel activity to a level 97.5±32.5% (n=6), 141.8±40.3% (n=8) and 221.6 ±75% (n=9) of that in the cell-attached mode, respectively. However, addition of high concentration of CaM (3.5 and 7.0μM) induced channel activity to a level lower than that of control conditions [64.6±28.3% (n=11) and 20.2±11.7% (n=7) respectively]. In order to further verify this effect, we carried out similar experiment on the same cell. At free [Ca²⁺] of 100 nM, perfusion of the patch with 2.1μM CaM + ATP obviously increased channel activity. However, the following application of 7.0μM CaM + ATP inhibited the reversed channel activity.2. Effect of CaM + ATP on Ca²⁺ channel activity is Ca²⁺-dependent.Same concentration of CaM had different effect on channel activity in inside-out mode when calcium concentration was different. When free [Ca²⁺] was 0, 250 and 500 nM, 1.4μM CaM induced channel activity to a level 89.6±21.0% (n=7), 219.8±71.1% (n=10) and 36.0±12.6% (n=7) of that in the cell-attached mode, respectively.3. Effect of CaM is not due to phosphorylation and dephosphorylation when the period of rundown is 1 minute.CaMKII inhibitor KN-62 (10μM) and the broad protein kinase inhibitor K252a (10μM) could not block the effect of CaM, suggesting that the effect of CaM was not mediated by phosphorylation by CaMKII and any other protein kinases. Ca²⁺ channel activity induced by 0.4μM CaM in excised patches at 2μM [Ca²⁺] was not affected by cyclosporin A (1μM), suggesting that the effect of CaM at high [Ca²⁺] did not correlate with dephosphorylation mediated by PP2B.4. Calcium concentration influences affinity of CaM for the channel.The results displayed a lower Kd value for facilitation than inactivation at certain [Ca²⁺]. And with the [Ca²⁺] increased, the Kd values both for CaM-induced facilitation and inactivation decreased. It means Ca²⁺ enhances the affinity of CaM for the channel. In addition, the result strongly supports the idea that there are two separate effector sites which interact with CaM inducing different modulation of the channel. 5. Effect of CaM₁₂₃₄ + ATP on Ca²⁺ channel activity in inside-out patch mode.When the excised patch was exposed to CaM₁₂₃₄ + ATP solutions containing different concentrations of free Ca²⁺, the channel activity induced by 3.5βM CAM₁₂₃₄ + ATP remained unchanged when [Ca²⁺] rose from zero to 250 nM. In accord with wild-type CaM, CAM₁₂₃₄ induced minimal channel activity when free [Ca²⁺] increased to micromolar levels. Furthermore, only the inactivation phase seemed to be shifted in a Ca²⁺-dependent manner. Thus, the significantly different recovered channel activity and the left shifting for CaM₁₂₃₄-induced inactivation phase at high [Ca²⁺] strongly support the concept that an unusual molecular mechanism of L-type Ca²⁺ channel regulation might persist at micromolar [Ca²⁺].6. Contour plot shows the complicated effects of CaM and Ca²⁺ on the channel activity.The constructed contour plot of the channel activity shows several features for the dependence of the channel activity on CaM and Ca²⁺. First, Ca²⁺ channel activity is only produced in a limited range of [CAM], approximately 0.1-10μM. The left and right sides of the peak effect of CaM represent the facilitation and inactivation phases, respectively. Second, Ca²⁺ shifts this 'CaM window' to the left (to lower concentrations). Third, at high [Ca²⁺], the channel activity is abolished at any [CAM].Conclusion1. CaM could directly induce facilitation and inactivation of Cavl.2 Ca²⁺ channel and this effect is Ca²⁺-dependent.2. Effect of Ca²⁺/CAM is not due to phosphorylation and dephosphorylation when the period of rundown is 1 minute.3. CaM is a crucial Ca²⁺ sensor for both facilitation and inactivation, and that mutant form CAM₁₂₃₄, which can not bind Ca²⁺ at any EF-hand, acts as dominant negatives to block both CDI and CDF.4. There are two distinct binding sites in N and/or C terminal of L-type channel responsible for Ca²⁺/CaM-dependent facilitation and inactivation, respectively.5. Ca²⁺ binding directly to EF hand motif plays an important role in the inactivation at micromolar Ca²⁺ concentration.