Nikolai M. Soldatov

Ca2+ sensors of L-type Ca2+ channel

Ca2+‐induced inactivation of L‐type Ca2+ is differentially mediated by two C‐terminal motifs of the α1C subunit, L (1572–1587) and K (1599–1651) implicated for calmodulin binding. We found that motif L is composed of a highly selective Ca2+ sensor and an adjacent Ca2+‐independent tethering site for calmodulin. The Ca2+ sensor contributes to higher Ca2+ sensitivity of the motif L complex with calmodulin. Since only combined mutation of both sites removes Ca2+‐dependent current decay, the two‐site modulation by Ca2+ and calmodulin may underlie Ca2+‐induced inactivation of the channel.

Ca2+ channel moving tail: link between Ca2+-induced inactivation and Ca2+ signal transduction

Ca(2+)-induced inactivation is an important property of L-type voltage-gated Ca(2+) channels. However, the underlying mechanisms are not yet understood well. There is general agreement that calmodulin (CaM) binds, in a Ca(2+)-dependent manner, to C-terminal motifs LA and IQ of the pore-forming alpha 1C-subunit and acts as a sensor that conveys Ca(2+)-induced inactivation. New data indicate that both Ca(2+)-induced inactivation and Ca(2+) signal transduction depend on the voltage-gated mobility of the C-terminal tail of the alpha 1C-subunit. It is proposed that LA is a Ca(2+)-sensitive lock for the mechanism of slow voltage-dependent inactivation of the channel. A Ca(2+)-dependent switch of CaM from LA to IQ removes CaM from the inner mouth of the pore and thus eliminates slow inactivation by facilitating the constriction of the pore. The mobile tail then shuttles the Ca(2+)-CaM-IQ complex to a downstream target of the Ca(2+) signaling cascade, where Ca(2+) is released as an activating stimulus. Apo-CaM rebinds to LA and returns to the pore for a new cycle of Ca(2+) signal transduction.

Atherosclerosis-related molecular alteration of the human CaV1.2 calcium channel α1C subunit

Atherosclerosis is an inflammatory process characterized by proliferation and dedifferentiation of vascular smooth muscle cells (VSMC). Cav1.2 calcium channels may have a role in atherosclerosis because they are essential for Ca2+-signal transduction in VSMC. The pore-forming Cav1.2α1 subunit of the channel is subject to alternative splicing. Here, we investigated whether the Cav1.2α1 splice variants are affected by atherosclerosis. VSMC were isolated by laser-capture microdissection from frozen sections of adjacent regions of arteries affected and not affected by atherosclerosis. In VSMC from nonatherosclerotic regions, RT-PCR analysis revealed an extended repertoire of Cav1.2α1 transcripts characterized by the presence of exons 21 and 41A. In VSMC affected by atherosclerosis, expression of the Cav1.2α1 transcript was reduced and the Cav1.2α1 splice variants were replaced with the unique exon-22 isoform lacking exon 41A. Molecular remodeling of the Cav1.2α1 subunits associated with atherosclerosis caused changes in electrophysiological properties of the channels, including the kinetics and voltage-dependence of inactivation, recovery from inactivation, and rundown of the Ca2+ current. Consistent with the pathophysiological state of VSMC in atherosclerosis, cell culture data pointed to a potentially important association of the exon-22 isoform of Cav1.2α1 with proliferation of VSMC. Our findings are consistent with a hypothesis that localized changes in cytokine expression generated by inflammation in atherosclerosis affect alternative splicing of the Cav1.2α1 gene in the human artery that causes molecular and electrophysiological remodeling of Cav1.2 calcium channels and possibly affects VSMC proliferation.

Differential Role of the α1C Subunit Tails in Regulation of the Cav1.2 Channel by Membrane Potential, β Subunits, and Ca2+ Ions

Voltage-gated Cav1.2 channels are composed of the pore-forming α1C and auxiliary β and α2δ subunits. Voltage-dependent conformational rearrangements of the α1C subunit C-tail have been implicated in Ca2+ signal transduction. In contrast, the α1C N-tail demonstrates limited voltage-gated mobility. We have asked whether these properties are critical for the channel function. Here we report that transient anchoring of the α1C subunit C-tail in the plasma membrane inhibits Ca2+-dependent and slow voltage-dependent inactivation. Both α2δ and β subunits remain essential for the functional channel. In contrast, if α1C subunits with are expressed α2δ but in the absence of a β subunit, plasma membrane anchoring of the α1C N terminus or its deletion inhibit both voltage- and Ca2+-dependent inactivation of the current. The following findings all corroborate the importance of the α1C N-tail/β interaction: (i) co-expression of β restores inactivation properties, (ii) release of the α1C N terminus inhibits the β-deficient channel, and (iii) voltage-gated mobility of the α1C N-tail vis à vis the plasma membrane is increased in the β-deficient (silent) channel. Together, these data argue that both the α1C N- and C-tails have important but different roles in the voltage- and Ca2+-dependent inactivation, as well as β subunit modulation of the channel. The α1C N-tail may have a role in the channel trafficking and is a target of the β subunit modulation. The β subunit facilitates voltage gating by competing with the N-tail and constraining its voltage-dependent rearrangements. Thus, cross-talk between the α1C C and N termini, β subunit, and the cytoplasmic pore region confers the multifactorial regulation of Cav1.2 channels.

Voltage-gated Mobility of the Ca2+ Channel Cytoplasmic Tails and Its Regulatory Role

Transient increase in intracellular free Ca2+ concentration generated by the voltage-gated Cav1.2 channels acts as an important intracellular signal. By using fluorescence resonance energy transfer combined with patch clamp in living cells, we present evidence for voltage-gated mobility of the cytoplasmic tails of the Cav1.2 channel and for its regulatory role in intracellular signaling. Anchoring of the C-terminal tail to the plasma membrane caused an inhibition of its state-dependent mobility, channel inactivation, and CREB-dependent transcription. Release of the tail restored these functions suggesting a direct role for voltage-gated mobility of the C-terminal tail in Ca2+ signaling.

Activation of protein kinase C augments T-type Ca2+ channel activity without changing channel surface density.

T‐type Ca2+ channels play essential roles in numerous cellular processes. Recently, we reported that phorbol‐12‐myristate‐13‐acetate (PMA) potently enhanced the current amplitude of Cav3.2 T‐type channels reconstituted in Xenopus oocytes. Here, we have compared PMA modulation of the activities of Cav3.1, Cav3.2 and Cav3.3 channels, and have investigated the underlying mechanism. PMA augmented the current amplitudes of the three T‐type channel isoforms, but the fold stimulations and time courses differed. The augmentation effects were not mimicked by 4α‐PMA, an inactive stereoisomer of PMA, but were abolished by preincubation with protein kinase C (PKC) inhibitors, indicating that PMA augmented T‐type channel currents via activation of oocyte PKC. The stimulation effect on Cav3.1 channel activity by PKC was mimicked by endothelin when endothelin receptor type A was coexpressed with Cav3.1 in the Xenopus oocyte system. Pharmacological studies combined with fluorescence imaging revealed that the surface density of Cav3.1 T‐type channels was not significantly changed by activation of PKC. The PKC effect on Cav3.1 was localized to the cytoplasmic II–III loop using chimeric channels with individual cytoplasmic loops of Cav3.1 replaced by those of Cav2.1.

Crosstalk Between Voltage-Independent Ca2+ Channels and L-Type Ca2+ Channels in A7r5 Vascular Smooth Muscle Cells at Elevated Intracellular pH: Evidence for Functional Coupling Between L-Type Ca2+ Channels and a 2-APB–Sensitive Cation Channel

Abstract— This study was designed to investigate the role of voltage-independent and voltage-dependent Ca2+ channels in the Ca2+ signaling associated with intracellular alkalinization in A7r5 vascular smooth muscle cells. Extracellular administration of ammonium chloride (20 mmol/L) resulted in elevation of intracellular pH and activation of a sustained Ca2+ entry that was inhibited by 2-amino-ethoxydiphenyl borate (2-APB, 200 &mgr;mol/L) but not by verapamil (10 &mgr;mol/L). Alkalosis-induced Ca2+ entry was mediated by a voltage-independent cation conductance that allowed permeation of Ca2+ (PCa/PNa ≈6), and was associated with inhibition of L-type Ca2+ currents. Alkalosis-induced inhibition of L-type Ca2+ currents was dependent on the presence of extracellular Ca2+ and was prevented by expression of a dominant-negative mutant of calmodulin. In the absence of extracellular Ca2+, with Ba2+ or Na+ as charge carrier, intracellular alkalosis failed to inhibit but potentiated L-type Ca2+ channel currents. Inhibition of Ca2+ currents through voltage-independent cation channels by 2-APB prevented alkalosis-induced inhibition of L-type Ca2+ currents. Similarly, 2-APB prevented vasopressin-induced activation of nonselective cation channels and inhibition of L-type Ca2+ currents. We suggest the existence of a pH-controlled Ca2+ entry pathway that governs the activity of smooth muscle L-type Ca2+ channels due to control of Ca2+/calmodulin-dependent negative feedback regulation. This Ca2+ entry pathway exhibits striking similarity with the pathway activated by stimulation of phospholipase-C–coupled receptors, and may involve a similar type of cation channel. We demonstrate for the first time the tight functional coupling between these voltage-independent Ca2+ channels and classical voltage-gated L-type Ca2+ channels.

Molecular Rearrangements of the Kv2.1 Potassium Channel Termini Associated with Voltage Gating

The voltage-gated Kv2.1 channel is composed of four identical subunits folded around the central pore and does not inactivate appreciably during short depolarizing pulses. To study voltage-induced relative molecular rearrangements of the channel, Kv2.1 subunits were genetically fused with enhanced cyan fluorescent protein and/or enhanced yellow fluorescent protein, expressed in COS1 cells, and investigated using fluorescence resonance energy transfer (FRET) microscopy combined with patch clamp. Fusion of fluorophores to either or both termini of the Kv2.1 monomer did not significantly affect the gating properties of the channel. FRET between the N- and C-terminal tags fused to the same or different Kv2.1 monomers decreased upon activation of the channel by depolarization from -80 to +60 mV, suggesting voltage-gated relative rearrangement between the termini. Because FRET between the Kv2.1 N- or C-terminal tags and the membrane-trapped EYFPN-PH pleckstrin homology domains did not change on depolarization, voltage-gated relative movements between the Kv2.1 termini occurred in a plane parallel to the plasma membrane, within a distance of 1-10 nm. FRET between the N-terminal tags did not change upon depolarization, indicating that the N termini do not rearrange relative to each other, but they could either move cooperatively with the Kv2.1 tetramer or not move at all. No FRET was detected between the C-terminal tags. Assuming their randomized orientation in the symmetrically arranged Kv2.1 subunits, C termini may move outwards in order to produce relative rearrangements between N and C termini upon depolarization.

Molecular determinant for run‐down of L‐type Ca2+ channels localized in the carboxyl terminus of the α1C subunit

1 The role of the sequence 1572‐1651 in the C‐terminal tail of the α1C subunit in run‐down of Ca2+ channels was studied by comparing functional properties of the conventional α1C,77 channel with those of three isoforms carrying alterations in this motif. 2 The pore‐forming α1C subunits were co‐expressed with α2δ and β2a subunits in HEK‐tsA201 cells, a subclone of the human embryonic kidney cell line, and studied by whole‐cell and single‐channel patch‐clamp techniques. 3 Replacement of amino acids 1572‐1651 in α1C,77 with 81 different amino acids leading to α1C,86 significantly altered run‐down behaviour. Run‐down of Ba2+ currents was rapid with α1C,77 channels, but was slow with α1C,86. 4 Transfer of the α1C,86 segments L (amino acids 1572‐1598) or K (amino acids 1595‐1652) into the α1C,77 channel yielded α1C,77L and α1C,77K channels, respectively, the run‐down of which resembled more that of α1C,77. These results demonstrate that a large stretch of sequence between residues 1572 and 1652 of α1C,86 renders Ca2+ channels markedly resistant to run‐down. 5 The protease inhibitor calpastatin added together with ATP was able to reverse the run‐down of α1C,77 channels. Calpastatin expression was demonstrated in the HEK‐tsA cells by Western blot analysis. 6 These results indicate a significant role of the C‐terminal sequence 1572‐1651 of the α1C subunit in run‐down of L‐type Ca2+ channels and suggest this sequence as a target site for a modulatory effect by endogenous calpastatin.

Modulation of Ca2+ signalling in rat atrial myocytes: possible role of the α1c carboxyl terminal

Ca2+ influx through L‐type Cav1.2 (α1c) Ca2+ channels is a critical step in the activation of cardiac ryanodine receptors (RyRs) and release of Ca2+ via Ca2+‐induced Ca2+ release(CICR). The released Ca2+, in turn, is the dominant determinant of inactivation of the Ca2+ current (ICa) and termination of release. Although Ca2+ cross‐signalling is mediated by high Ca2+ fluxes in the microdomains of α1c‐RyR complexes, ICa‐gated Ca2+ cross‐signalling is surprisingly resistant to intracellular Ca2+ buffering and has steeply voltage‐dependent gain, inconsistent with a strict CICR mechanism, suggesting the existence of additional regulatory step(s). To explore the possible regulatory role of the carboxyl (C)‐terminal tail of α1c in modulating Ca2+ signalling, we tested the effects of introducing two α1c C‐terminal peptides, LA (1571–1599) and K (1617–1636) on the central α1c‐unassociated Ca2+‐release sites of atrial myocytes, using rapid (240 Hz) two‐dimensional confocal Ca2+ imaging. The frequency of spontaneously activating central sparks increased by approximately fourfold on dialysing LA‐ but not K‐peptide into myocytes voltage‐clamped at ‐80 mV. The rate but not the magnitude of caffeine (10 mM)‐triggered central Ca2+ release was significantly accelerated by LA‐ but not K‐peptide. Individual Ca2+ spark size and flux were larger in LA‐ but not in K‐peptide‐dialysed myocytes. Although LA‐peptide did not change the amplitude or inactivation kinetics of ICa, LA‐peptide did strongly enhance the central Ca2+ transients triggered by ICa at ‐30 mV (small ICa) but not at +20 mV (large ICa). In contrast, K‐peptide had no effect on either ICa or the local Ca2+ transients. LA‐peptide with a deleted calmodulin‐binding region (LM1‐peptide) had no significant effects on the central spark frequency but suppressed spontaneous spark frequency in the periphery. Our results indicate that the calmodulin‐binding LA motif of the α1c C‐terminal tail may sensitize the RyRs, thereby increasing their open probability and providing for both the voltage‐dependence of CICR and the higher frequency of spark occurrence in the periphery of atrial myocytes where the native α1c‐RyR complexes are intact.