Igor Dzhura

Calmodulin kinase determines calcium-dependent facilitation of L-type calcium channels

A dynamic positive feedback mechanism, known as ‘facilitation’, augments L-type calcium-ion currents (ICa) in response to increased intracellular Ca2+ concentrations. The Ca2+-binding protein calmodulin (CaM) has been implicated in facilitation, but the single-channel signature and the signalling events underlying Ca2+/CaM-dependent facilitation are unknown. Here we show that the Ca2+/CaM-dependent protein kinase II (CaMK) is necessary and possibly sufficient for ICa facilitation. CaMK induces a channel-gating mode that is characterized by frequent, long openings of L-type Ca2+ channels. We conclude that CaMK-mediated phosphorylation is an essential signalling event in triggering Ca2+/CaM-dependent ICa facilitation.

Calmodulin Kinase II and Arrhythmias in a Mouse Model of Cardiac Hypertrophy

Background—Calmodulin kinase (CaMK) II is linked to arrhythmia mechanisms in cellular models where repolarization is prolonged. CaMKII upregulation and prolonged repolarization are general features of cardiomyopathy, but the role of CaMKII in arrhythmias in cardiomyopathy is unknown. Methods and Results—We studied a mouse model of cardiac hypertrophy attributable to transgenic (TG) overexpression of a constitutively active form of CaMKIV that also has increased endogenous CaMKII activity. ECG-telemetered TG mice had significantly more arrhythmias than wild-type (WT) littermate controls at baseline, and arrhythmias were additionally increased by isoproterenol. Arrhythmias were significantly suppressed by an inhibitory agent targeting endogenous CaMKII. TG mice had longer QT intervals and action potential durations than WT mice, and TG cardiomyocytes had frequent early afterdepolarizations (EADs), a hypothesized mechanism for triggering arrhythmias. EADs were absent in WT cells before and after isoproterenol, whereas EAD frequency was unaffected by isoproterenol in TG mice. L-type Ca2+ channels (LTTCs) can activate EADs, and LTCC opening probability (Po) was significantly higher in TG than WT cardiomyocytes before and after isoproterenol. A CaMKII inhibitory peptide equalized TG and WT LTCC Po and eliminated EADs, whereas a peptide antagonist of the Na+/Ca2+ exchanger current, also hypothesized to support EADs, was ineffective. Conclusions—These findings support the hypothesis that CaMKII is a proarrhythmic signaling molecule in cardiac hypertrophy in vivo. Cellular studies point to EADs as a triggering mechanism for arrhythmias but suggest that the increase in arrhythmias after &bgr;-adrenergic stimulation is independent of enhanced EAD frequency.

Calmodulin kinase and a calmodulin-binding ‘IQ’ domain facilitate L-type Ca2+ current in rabbit ventricular myocytes by a common mechanism

1 Ca2+‐calmodulin‐dependent protein kinase II (CaMK) and a calmodulin (CaM)‐binding ‘IQ’ domain (IQ) are both implicated in Ca2+‐dependent regulation of L‐type Ca2+ current (ICa). We used an IQ‐mimetic peptide (IQmp), under conditions in which CaMK activity was controlled, to test the relationship between these CaM‐activated signalling elements in the regulation of L‐type Ca2+ channels (LTCCs) and ICa in rabbit ventricular myocytes. 2 A specific CaMK inhibitory peptide nearly abolished ICa facilitation, but the facilitation was ‘rescued’ by cell dialysis with IQmp. 3 IQmp significantly enhanced ICa facilitation and slowed the fast component of ICa inactivation, compared with an inactive control peptide. Neither effect could be elicited by a more avid CaM‐binding peptide, suggesting that generalized CaM buffering did not account for the effects of IQmp. 4 I Ca facilitation was abolished and the fast component of inactivation eliminated by ryanodine, caffeine or thapsigargin, suggesting that the sarcoplasmic reticulum (SR) is an important source of Ca2+ for ICa facilitation and inactivation. IQmp did not restore ICa facilitation under these conditions. 5 Engineered Ca2+‐independent CaMK and IQmp each markedly increased LTCC open probability (Po) in excised cell membrane patches. The LTCC Po increases with CaMK and IQmp were non‐additive, suggesting that CaMK and IQmp are components of a shared signalling pathway. 6 Both CaMK and IQmp induced a modal gating shift in LTCCs that favoured prolonged openings, indicating that CaMK and IQmp affect LTCCs through a common biophysical mechanism. 7 These findings support the hypothesis that CaMK is required for physiological ICa facilitation in cardiac myocytes. Both CaMK and IQmp were able to induce a modal gating shift in LTCCs, suggesting that each of these signalling elements is important for Ca2+‐CaM‐dependent LTCC facilitation in cardiac myocytes.

Cytoskeletal disrupting agents prevent calmodulin kinase, iq domain and voltage‐dependent facilitation of l‐type ca2+ Channels

A calmodulin (CaM) binding ‘IQ’ domain on the L‐type Ca2+ channel (LTCC) C terminus and calmodulin kinase II (CaMK) both signal increases in LTCC opening probability (Po) by shifting LTCCs into a gating mode (mode 2) with long openings through a process called facilitation. However, the mechanism whereby CaMK and the IQ domain are targeted to LTCCs is unknown. Endogenous CaMK is targeted to LTCCs in excised cell membrane patches because LTCC Po increased significantly in CaM‐enriched (20 μm) bath solution and this effect was prevented by a specific CaMK inhibitory peptide, but not by an inactive control peptide. Pre‐exposure of myocytes to the cytoskeletal disrupting agents nocodazole (microtubule specific) or cytochalasin D (microfilament specific) prevented the effects of CaM‐dependent increases in Po of LTCCs in excised membrane patches. Neither cytochalasin D nor nocodazole altered the distribution of LTCC gating modes under basal conditions in on‐cell mode or excised cell membrane patches, but each of these agents occluded the response of LTCCs to exogenous, constitutively active CaMK and to an IQ‐mimetic peptide (IQmp). Cytochalasin D and nocodazole pretreatment also prevented LTCC facilitation that followed a cell membrane depolarizing prepulse. In contrast, cytochalasin D and nocodazole did not affect the increase in LTCC Po or prevent the shift to mode 2 gating in response to protein kinase A, indicating that cytoskeletal disruption specifically prevents prepulse, CaMK and IQ‐dependent LTCC facilitation.

A dynamic α-β inter-subunit agonist signaling complex is a novel feedback mechanism for regulating L-type Ca2+ channel opening

L‐type Ca2+ channels are macromolecular protein complexes in neurons and myocytes that open in response to cell membrane depolarization to supply Ca2+ for regulating gene transcription and vesicle secretion and triggering cell contraction. L‐type Ca2+ channels include a pore‐forming α and an auxiliary β subunit, and α subunit openings are regulated by cellular Ca2+ through a mechanism involving the Ca2+‐sensing protein calmodulin (CaM) and CaM binding motifs in the α subunit cytoplasmic C terminus. Here we show that these CaM binding motifs are “autoagonists” that increase α subunit openings by binding the β subunit. The CaM binding domains are necessary and sufficient for the α subunit C terminus to bind the β subunit in vitro, and excess CaM blocks this interaction. Addition of CaM binding domains to native cardiac L‐type Ca2+ channels in excised cell membrane patches increases openings, and this agonist effect is prevented by excess CaM. Recombinant LTCC openings are also increased by exogenous CaM binding domains by a mechanism requiring the β subunit, and excess CaM blocks this effect. Thus, the bifunctional ability of the α subunit CaM binding motifs to competitively associate with the β subunit or CaM provides a novel paradigm for feedback control of cellular Ca2+ entry.

C terminus L-type Ca2+ channel calmodulin-binding domains are 'auto-agonist' ligands in rabbit ventricular myocytes

L‐type Ca2+ channel C terminus calmodulin (CaM)‐binding domains are molecular determinants for Ca2+–CaM‐dependent increases in L‐type Ca2+ current (ICa), and a CaM‐binding IQ domain mimetic peptide (IQmp) increases L‐type Ca2+ channel current by promoting a gating mode with prolonged openings (mode 2), suggesting the intriguing possibility that CaM‐binding domains are ‘auto‐agonist’ signalling molecules. In order to test the breadth of this concept, we studied the effect of a second C terminus CaM‐binding domain (CB) mp (CBmp), in conjunction with IQmp, on single L‐type Ca2+ channel currents in excised cell membrane patches from rabbit ventricular myocytes. Here we show that both CBmp and IQmp are agonist ligands that non‐additively increase L‐type Ca2+ channel opening probability (Po) by inducing mode 2 gating. CBmp and IQmp agonist effects were lost under conditions favouring calcification of CaM (Ca2+–CaM, 150 nm free Ca2+ and 10–20 μm CaM), but persisted in the presence of CaM (0–20 μm) under conditions adverse to Ca2+–CaM (20 mm BAPTA), indicating that CaM‐binding domains increase L‐type Ca2+ channel Po by a low Ca2+–CaM activity mechanism. Increasing Ca2+–CaM in the bath (cytosol) reduced the efficacy of CBmp and IQmp signals with Ba2+ as charge carrier, suggesting that CaM binding motifs target a site outside of the pore region. We measured the combined effects of CBmp and Ca2+–CaM‐dependent protein kinase II (CaMKII) on L‐type Ca2+ channels by using an engineered Ca2+–CaM‐independent form of CaMKII that remains active under low Ca2+–CaM conditions, permissive for CBmp signalling. CBmp and CaMKII increased L‐type Ca2+ channel Po in a non‐additive manner, suggesting that low and high Ca2+–CaM‐dependent L‐type Ca2+ channel facilitation pathways converge upon a common signalling mechanism.

Differential effects of phospholamban and Ca2+/calmodulin-dependent kinase II on [Ca2+]i transients in cardiac myocytes at physiological stimulation frequencies

In cardiac myocytes, the activity of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is hypothesized to regulate Ca(2+) release from and Ca(2+) uptake into the sarcoplasmic reticulum via the phosphorylation of the ryanodine receptor 2 and phospholamban (PLN), respectively. We tested the role of CaMKII and PLN on the frequency adaptation of cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients in nearly 500 isolated cardiac myocytes from transgenic mice chronically expressing a specific CaMKII inhibitor, interbred into wild-type or PLN null backgrounds under physiologically relevant pacing conditions (frequencies from 0.2 to 10 Hz and at 37 degrees C). When compared with that of mice lacking PLN only, the combined chronic CaMKII inhibition and PLN ablation decreased the maximum Ca(2+) release rate by more than 50% at 10 Hz. Although PLN ablation increased the rate of Ca(2+) uptake at all frequencies, its combination with CaMKII inhibition did not prevent a frequency-dependent reduction of the amplitude and the duration of the [Ca(2+)](i) transient. High stimulation frequencies in the physiological range diminished the effects of PLN ablation on the decay time constant and on the maximum decay rate of the [Ca(2+)](i) transient, indicating that the PLN-mediated feedback on [Ca(2+)](i) removal is limited by high stimulation frequencies. Taken together, our results suggest that in isolated mouse ventricular cardiac myocytes, the combined chronic CaMKII inhibition and PLN ablation slowed Ca(2+) release at physiological frequencies: the frequency-dependent decay of the amplitude and shortening of the [Ca(2+)](i) transient occurs independent of chronic CaMKII inhibition and PLN ablation, and the PLN-mediated regulation of Ca(2+) uptake is diminished at higher stimulation frequencies within the physiological range.

Retraction: Calmodulin kinase determines calcium-dependent facilitation of L-type calcium channels

Retraction: Calmodulin kinase determines calcium-dependent facilitation of L-type calcium channels

Erratum: Calmodulin kinase determines calcium-dependent facilitation of L-type calcium channels (Nature Cell Biology (2000) 2 (173-177))

Retraction: Calmodulin kinase determines calcium-dependent facilitation of L-type calcium channels

Retraction Notice To: L-Type Ca2+ Channel Facilitation Mediated by Phosphorylation of the β Subunit by CaMKII

(Molecular Cell 23, 641–650; September 1, 2006)This article has been retracted at the request of the authors. The NIH Office of Research Integrity (ORI) has investigated the third author of this paper, Igor Dzhura. Following the investigation, the ORI reported that Dzhura “engaged in research misconduct by submitting and publishing multiple falsified and/or fabricated action potential traces” in research he conducted in multiple laboratories (published November 2014; https://ori.hhs.gov/content/case-summary-dzhura-igor).Dzhura’s sole contribution to this paper was the data reported in Figure 3, showing single-channel activities of CaV1.2 L-type calcium channels under various conditions. Inspection of Figure 3 revealed inappropriate duplications of exemplar current traces. While subsequent published studies have supported the central findings of this paper, including data showing that CaMKII facilitates CaV1.2 channels in cardiomyocytes by binding to and phosphorylating Thr498 in the β2a subunit (Koval et al. [2010], Proc. Natl. Acad. Sci. USA 107, 4996–5000), the authors are retracting the paper because it contains falsified data. The authors apologize to the scientific community for any inconveniences or challenges resulting from the publication and retraction of this manuscript.