RyRCa2+ Leak Limits Cardiac Ca2+ Window Current Overcoming the Tonic Effect of Calmodulin in Mice

Ca2+ mediates the functional coupling between L-type Ca2+ channel (LTCC) and sarcoplasmic reticulum (SR) Ca2+ release channel (ryanodine receptor, RyR), participating in key pathophysiological processes. This crosstalk manifests as the orthograde Ca2+-induced Ca2+-release (CICR) mechanism triggered by Ca2+ influx, but also as the retrograde Ca2+-dependent inactivation (CDI) of LTCC, which depends on both Ca2+ permeating through the LTCC itself and on SR Ca2+ release through the RyR. This latter effect has been suggested to rely on local rather than global Ca2+ signaling, which might parallel the nanodomain control of CDI carried out through calmodulin (CaM). Analyzing the CICR in catecholaminergic polymorphic ventricular tachycardia (CPVT) mice as a model of RyR-generated Ca2+ leak, we evidence here that increased occurrence of the discrete local SR Ca2+ releases through the RyRs (Ca2+ sparks) causea depolarizing shift in activation and a hyperpolarizing shift inisochronic inactivation of cardiac LTCC current resulting in the reduction of window current. Both increasing fast [Ca2+]i buffer capacity or depleting SR Ca2+ store blunted these changes, which could be reproduced in WT cells by RyRCa2+ leak induced with Ryanodol and CaM inhibition.Our results unveiled a new paradigm for CaM-dependent effect on LTCC gating and further the nanodomain Ca2+ control of LTCC, emphasizing the importance of spatio-temporal relationships between Ca2+ signals and CaM function

Channelopathies in Cav1.1, Cav1.3, and Cav1.4 voltage-gated L-type Ca2+ channels

Voltage-gated Ca2+ channels couple membrane depolarization to Ca2+-dependent intracellular signaling events. This is achieved by mediating Ca2+ ion influx or by direct conformational coupling to intracellular Ca2+ release channels. The family of Cav1 channels, also termed L-type Ca2+ channels (LTCCs), is uniquely sensitive to organic Ca2+ channel blockers and expressed in many electrically excitable tissues. In this review, we summarize the role of LTCCs for human diseases caused by genetic Ca2+ channel defects (channelopathies). LTCC dysfunction can result from structural aberrations within their pore-forming α1 subunits causing hypokalemic periodic paralysis and malignant hyperthermia sensitivity (Cav1.1 α1), incomplete congenital stationary night blindness (CSNB2; Cav1.4 α1), and Timothy syndrome (Cav1.2 α1; reviewed separately in this issue). Cav1.3 α1 mutations have not been reported yet in humans, but channel loss of function would likely affect sinoatrial node function and hearing. Studies in mice revealed that LTCCs indirectly also contribute to neurological symptoms in Ca2+ channelopathies affecting non-LTCCs, such as Cav2.1 α1 in tottering mice. Ca2+ channelopathies provide exciting disease-related molecular detail that led to important novel insight not only into disease pathophysiology but also to mechanisms of channel function

Developmental activation of calmodulin-dependent facilitation of cerebellar P-type Ca2+current

10.1523/JNEUROSCI.2253-05.2005Journal of Neuroscience25368282-829

Novel functional properties of Ca2+ channel β subunits revealed by their expression in adult rat heart cells

Recombinant adenoviruses were used to overexpress green fluorescent protein (GFP)-fused auxiliary Ca2+ channel β subunits (β1-β4) in cultured adult rat heart cells, to explore new dimensions of β subunit functions in vivo. Distinct β-GFP subunits distributed differentially between the surface sarcolemma, transverse elements, and nucleus in single heart cells. All β-GFP subunits increased the native cardiac whole-cell L-type Ca2+ channel current density, but produced distinctive effects on channel inactivation kinetics. The degree of enhancement of whole-cell current density was non-uniform between β subunits, with a rank order of potency β2a α β4 > β1b > β3. For each β subunit, the increase in L-type current density was accompanied by a correlative increase in the maximal gating charge (Qmax) moved with depolarization. However, β subunits produced characteristic effects on single L-type channel gating, resulting in divergent effects on channel open probability (Po). Quantitative analysis and modelling of single-channel data provided a kinetic signature for each channel type. Spurred on by ambiguities regarding the molecular identity of the actual endogenous cardiac L-type channel β subunit, we cloned a new rat β2 splice variant, β2b, from heart using 5′ rapid amplification of cDNA ends (RACE) PCR. By contrast with β2a, expression of β2b in heart cells yielded channels with a microscopic gating signature virtually identical to that of native unmodified channels. Our results provide novel insights into β subunit functions that are unattainable in traditional heterologous expression studies, and also provide new perspectives on the molecular identity of the β subunit component of cardiac L-type Ca2+ channels. Overall, the work establishes a powerful experimental paradigm to explore novel functions of ion channel subunits in their native environments

Calmodulin kinase II accelerates L-type Ca(2+) current recovery from inactivation and compensates for the direct inhibitory effect of [Ca(2+)](i) in rat ventricular myocytes

Some studies report that the positive relationship between L-type Ca(2+) current (I(Ca−L)) and frequency in cardiac myocytes is mainly due to a direct negative feedback of sarcoplasmic reticulum Ca(2+) release on I(Ca−L) inactivation while others provide evidence for activation of calmodulin kinase II (CaMKII). To further elucidate the role of endogenous CaMKII activity, the CaMKII inhibitory peptides, autocamtide-2 relating inhibitory peptide (AIP) and myristoylated AIP were applied using conventional and perforated patch-clamp methods. AIP inhibited the normal adaptive increase in I(Ca−L) in response to abrupt increase in pacing frequency from 0.05 to 2 Hz. The positive I(Ca−L)–frequency relationship was reversed by AIP and the inhibitory effect of AIP was significantly exaggerated at fast pacing rates. The onset of inactivation of I(Ca−L) was not altered by AIP. After thapsigargin treatment, AIP slowed recovery from inactivation of I(Ca−L) and this effect was exaggerated during fast pacing. Buffering of [Ca(2+)](i) by BAPTA and EGTA accelerated recovery of I(Ca−L) from inactivation, and BAPTA partly eliminated the effect of AIP on the recovery. We conclude that: (1) [Ca(2+)](i) directly slows I(Ca−L) recovery from inactivation; and (2) Ca(2+)-dependent endogenous CaMKII activity accelerates the I(Ca−L) recovery. Thus, at fast heart rates, elevated [Ca(2+)](i) activates endogenous CaMKII and compensates for its direct inhibitory effect on I(Ca−L) recovery from inactivation. Dynamic activity of endogenous CaMKII enhances the positive I(Ca−L)–frequency relationship