Duane D. Hall

Supramolecular Assemblies and Localized Regulation of Voltage-Gated Ion Channels

This review addresses the localized regulation of voltage-gated ion channels by phosphorylation. Comprehensive data on channel regulation by associated protein kinases, phosphatases, and related regulatory proteins are mainly available for voltage-gated Ca2+ channels, which form the main focus of this review. Other voltage-gated ion channels and especially Kv7.1-3 (KCNQ1-3), the large- and small-conductance Ca2+-activated K+ channels BK and SK2, and the inward-rectifying K+ channels Kir3 have also been studied to quite some extent and will be included. Regulation of the L-type Ca2+ channel Cav1.2 by PKA has been studied most thoroughly as it underlies the cardiac fight-or-flight response. A prototypical Cav1.2 signaling complex containing the beta2 adrenergic receptor, the heterotrimeric G protein Gs, adenylyl cyclase, and PKA has been identified that supports highly localized via cAMP. The type 2 ryanodine receptor as well as AMPA- and NMDA-type glutamate receptors are in close proximity to Cav1.2 in cardiomyocytes and neurons, respectively, yet independently anchor PKA, CaMKII, and the serine/threonine phosphatases PP1, PP2A, and PP2B, as is discussed in detail. Descriptions of the structural and functional aspects of the interactions of PKA, PKC, CaMKII, Src, and various phosphatases with Cav1.2 will include comparisons with analogous interactions with other channels such as the ryanodine receptor or ionotropic glutamate receptors. Regulation of Na+ and K+ channel phosphorylation complexes will be discussed in separate papers. This review is thus intended for readers interested in ion channel regulation or in localization of kinases, phosphatases, and their upstream regulators.

CaMKII determines mitochondrial stress responses in heart

Myocardial cell death is initiated by excessive mitochondrial Ca2+ entry causing Ca2+ overload, mitochondrial permeability transition pore (mPTP) opening and dissipation of the mitochondrial inner membrane potential (ΔΨm). However, the signalling pathways that control mitochondrial Ca2+ entry through the inner membrane mitochondrial Ca2+ uniporter (MCU) are not known. The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated in ischaemia reperfusion, myocardial infarction and neurohumoral injury, common causes of myocardial death and heart failure; these findings suggest that CaMKII could couple disease stress to mitochondrial injury. Here we show that CaMKII promotes mPTP opening and myocardial death by increasing MCU current (IMCU). Mitochondrial-targeted CaMKII inhibitory protein or cyclosporin A, an mPTP antagonist with clinical efficacy in ischaemia reperfusion injury, equivalently prevent mPTP opening, ΔΨm deterioration and diminish mitochondrial disruption and programmed cell death in response to ischaemia reperfusion injury. Mice with myocardial and mitochondrial-targeted CaMKII inhibition have reduced IMCU and are resistant to ischaemia reperfusion injury, myocardial infarction and neurohumoral injury, suggesting that pathological actions of CaMKII are substantially mediated by increasing IMCU. Our findings identify CaMKII activity as a central mechanism for mitochondrial Ca2+ entry in myocardial cell death, and indicate that mitochondrial-targeted CaMKII inhibition could prevent or reduce myocardial death and heart failure in response to common experimental forms of pathophysiological stress.

Age-dependent requirement of AKAP150-anchored PKA and GluR2-lacking AMPA receptors in LTP

Association of PKA with the AMPA receptor GluR1 subunit via the A kinase anchor protein AKAP150 is crucial for GluR1 phosphorylation. Mutating the AKAP150 gene to specifically prevent PKA binding reduced PKA within postsynaptic densities (>70%). It abolished hippocampal LTP in 7–12 but not 4‐week‐old mice. Inhibitors of PKA and of GluR2‐lacking AMPA receptors blocked single tetanus LTP in hippocampal slices of 8 but not 4‐week‐old WT mice. Inhibitors of GluR2‐lacking AMPA receptors also prevented LTP in 2 but not 3‐week‐old mice. Other studies demonstrate that GluR1 homomeric AMPA receptors are the main GluR2‐lacking AMPA receptors in adult hippocampus and require PKA for their functional postsynaptic expression during potentiation. AKAP150‐anchored PKA might thus critically contribute to LTP in adult hippocampus in part by phosphorylating GluR1 to foster postsynaptic accumulation of homomeric GluR1 AMPA receptors during initial LTP in 8‐week‐old mice.

Regulation of the Cln3–Cdc28 kinase by cAMP in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae grows at widely varying rates in different growth media. In order to maintain a relatively constant cell size, yeast cells must regulate the rate of progress through the cell cycle to match changes in growth rate, moving quickly through G1 in rich medium, and slowly in poor medium. We have examined connections between nutrients, and the expression and activity of Cln3–Cdc28 kinase that regulates the G1–S boundary of the cell cycle in yeast, a point referred to as Start. We find that Cln3 protein levels are highest in glucose and lower in poorer carbon sources. This regulation involves both transcriptional and post‐transcriptional control. Although the Ras–cAMP pathway does not appear to affect CLN3 transcription, cAMP increases Cln3 protein levels and Cln3–Cdc28 kinase activity. This regulation requires untranslated regions of the CLN3 message, and can be explained by changes in protein synthesis rates caused by cAMP. A model for CLN3 regulation and function is presented in which CLN3 regulates G1 length in response to nutrients.

Physical and Functional Interaction Between Calcineurin and the Cardiac L-Type Ca2+ Channel

The L-type Ca2+ channel (LTCC) is the major mediator of Ca2+ influx in cardiomyocytes, leading to both mechanical contraction and activation of signaling cascades. Among these Ca2+-activated cascades is calcineurin, a protein phosphatase that promotes hypertrophic growth of the heart. Coimmunoprecipitations from heart extracts and pulldowns using heterologously expressed proteins provided evidence for direct binding of calcineurin at both the N and C termini of α11.2. At the C terminus, calcineurin bound specifically at amino acids 1943 to 1971, adjacent to a well-characterized protein kinase (PK)A/PKC/PKG phospho-acceptor site Ser1928. In vitro assays demonstrated that calcineurin can dephosphorylate α11.2. Channel function was increased in voltage-clamp recordings of ICa,L from cultured cardiomyocytes expressing constitutively active calcineurin, consistent with previous observations in cardiac hypertrophy in vivo. Conversely, acute suppression of calcineurin pharmacologically or with specific peptides decreased ICa,L. These data reveal direct physical interaction between the LTCC and calcineurin in heart. Furthermore, they demonstrate that calcineurin induces robust increases in ICa,L and highlight calcineurin as a key modulator of pathological electrical remodeling in cardiac hypertrophy.

Assembly of a β2‐adrenergic receptor—GluR1 signalling complex for localized cAMP signalling

Central noradrenergic signalling mediates arousal and facilitates learning through unknown molecular mechanisms. Here, we show that the β2‐adrenergic receptor (β2AR), the trimeric Gs protein, adenylyl cyclase, and PKA form a signalling complex with the AMPA‐type glutamate receptor subunit GluR1, which is linked to the β2AR through stargazin and PSD‐95 and their homologues. Only GluR1 associated with the β2AR is phosphorylated by PKA on β2AR stimulation. Peptides that interfere with the β2AR–GluR1 association prevent this phosphorylation of GluR1. This phosphorylation increases GluR1 surface expression at postsynaptic sites and amplitudes of EPSCs and mEPSCs in prefrontal cortex slices. Assembly of all proteins involved in the classic β2AR–cAMP cascade into a supramolecular signalling complex and thus allows highly localized and selective regulation of one of its major target proteins.

The mitochondrial uniporter controls fight or flight heart rate increases

Heart rate increases are a fundamental adaptation to physiological stress, while inappropriate heart rate increases are resistant to current therapies. However, the metabolic mechanisms driving heart rate acceleration in cardiac pacemaker cells remain incompletely understood. The mitochondrial calcium uniporter (MCU) facilitates calcium entry into the mitochondrial matrix to stimulate metabolism. We developed mice with myocardial MCU inhibition by transgenic expression of a dominant negative (DN) MCU. Here we show that DN-MCU mice had normal resting heart rates but were incapable of physiological fight or flight heart rate acceleration. We found MCU function was essential for rapidly increasing mitochondrial calcium in pacemaker cells and that MCU enhanced oxidative phoshorylation was required to accelerate reloading of an intracellular calcium compartment prior to each heartbeat. Our findings show the MCU is necessary for complete physiological heart rate acceleration and suggest MCU inhibition could reduce inappropriate heart rate increases without affecting resting heart rate.

AKAP150-anchored PKA activity is important for LTD during its induction phase

Protein kinase A (PKA) is thought to tonically maintain an enhanced level of postsynaptic AMPA receptor responses. Injection of PKA inhibitory peptides leads to a run‐down of AMPA receptor responses and prevents long‐term depression (LTD). This run‐down of AMPA receptor activity was proposed to occlude a further reduction that would otherwise constitute LTD. PKA is recruited to postsynaptic sites by the A kinase anchor protein AKAP150. We found that LTD was strongly impaired in acute hippocampal slices from 2‐week‐old mice in which the PKA binding site on AKAP150 had been genetically deleted (D36 mice). However, basal postsynaptic AMPA and NMDA receptor activity was indistinguishable between D36 and WT mice. During extracellular recordings of field EPSPs and during intracellular recording of EPSCs from hippocampal slices from WT mice, H‐89 and KT5720, two structurally different PKA inhibitors, inhibited LTD by more than 70% without affecting basal synaptic transmission or basal phosphorylation of serine 845 on GluR1. Collectively our data indicate that AKAP150‐anchored PKA activity is required to induce LTD and not merely to maintain a tonically heightened activity level of AMPA receptors as proposed earlier.

Competition between α-actinin and Ca2+-Calmodulin Controls Surface Retention of the L-type Ca2+ Channel CaV1.2

Regulation of neuronal excitability and cardiac excitation-contraction coupling requires the proper localization of L-type Ca²⁺ channels. We show that the actin-binding protein α-actinin binds to the C-terminal surface targeting motif of α11.2, the central pore-forming Ca(V)1.2 subunit, in order to foster its surface expression. Disruption of α-actinin function by dominant-negative or small hairpin RNA constructs reduces Ca(V)1.2 surface localization in human embryonic kidney 293 and neuronal cultures and dendritic spine localization in neurons. We demonstrate that calmodulin displaces α-actinin from their shared binding site on α11.2 upon Ca²⁺ influx through L-type channels, but not through NMDAR, thereby triggering loss of Ca(V)1.2 from spines. Coexpression of a Ca²⁺-binding-deficient calmodulin mutant does not affect basal Ca(V)1.2 surface expression but inhibits its internalization upon Ca²⁺ influx. We conclude that α-actinin stabilizes Ca(V)1.2 at the plasma membrane and that its displacement by Ca²⁺-calmodulin triggers Ca²⁺-induced endocytosis of Ca(V)1.2, thus providing an important negative feedback mechanism for Ca²⁺ influx.

AZF1 Is a Glucose-Dependent Positive Regulator of CLN3 Transcription in Saccharomyces cerevisiae

ABSTRACT Transcription of the CLN3 G1 cyclin in Saccharomyces cerevisiae is positively regulated by glucose in a process that involves a set of DNA elements with the sequence AAGAAAAA (A2GA5). To identify proteins that interact with these elements, we used a 1-hybrid approach, which yielded a nuclear zinc finger protein previously identified as Azf1. Gel shift and chromatin immunoprecipitation experiments show that Azf1 binds to the A2GA5 CLN3 regulatory sequences in vitro and in vivo, thus identifying a transcriptional regulatory protein for CLN3 and a DNA sequence target for Azf1. We show that glucose-induced expression of a reporter gene driven by the A2GA5 CLN3 regulatory sequences is dependent upon the presence of AZF1. Furthermore, deletion of AZF1 markedly reduces the transcriptional induction of CLN3 by glucose. In addition, Azf1 can induce reporter expression in a glucose-specific manner when artificially tethered to a promoter via the DNA-binding domain from Gal4. We conclude that AZF1 is a glucose-dependent transcription factor that interacts with the CLN3 A2GA5 repeats to play a positive role in the regulation of CLN3 mRNA expression by glucose.