Geoffrey S. Pitt and Steven O. Marx

Chapter Outline

Ca²⁺: The Final Signal of Electrical Activity and Regulator of Ion Channels

In adult cardiomyocytes, voltage-gated ion channels exist solely to change intracellular Ca2⁺. Whether regulating action potentials in myocytes, neurons, or hormone-secreting cells, ion channels control membrane voltage and thereby either activate or inhibit voltage-gated Ca²⁺ channels to control Ca²⁺ entry into the cells. K⁺ channels can control resting membrane potential and drive action potential repolarization, but they do so to limit Ca²⁺ entry through L-type Ca_V1.2 Ca²⁺ channels. Voltage-gated Na⁺ channels could be essential for the initiation of action potentials in the working myocardium, but the net effect of action potentials is to open Ca_V1.2 Ca²⁺ channels to translate electrical activity into cellular contraction. Not only does Ca²⁺ serve as the final signal of cellular electrical activity, but Ca²⁺ also actively participates in the regulation of many of the channels responsible for controlling the cardiac action potential and thereby for controlling Ca_V1.2 gating and the consequent intracellular Ca²⁺ signal. Growing evidence over the past decade has demonstrated that Ca²⁺ can regulate cardiac ion channels at many levels. For example, Ca²⁺ can control channel transcription, biosynthesis, trafficking, or gating of mature channels at the sarcolemma.

Unique Features of Ca²⁺ as a Signaling Ion in Myocytes

The efficacy of Ca²⁺ as an intracellular signal derives from specific properties that set it apart from other intracellular ions. First, Ca²⁺ signals have a broad dynamic range. Global intracellular Ca²⁺ can rapidly increase more than tenfold, and even more in certain subcellular locations such as near the mouth of an ion channel. This feature results from the concerted actions of several cellular mechanisms designed to keep free cytoplasmic Ca²⁺ concentrations low and thereby prevent precipitation with organic phosphates, a major intracellular counter anion. These Ca²⁺-limiting mechanisms maintain a free intracellular Ca²⁺ concentration ([Ca²⁺]_i) close to 100 nM during diastole, which rises to upwards of 1 µM during the plateau phase of the action potential, in contrast to an extracellular free Ca²⁺ concentration of 1 to 2 mM. Thus, there is a strong chemical driving force for Ca²⁺ entry. Second, Ca²⁺ entry is fast. When Ca_V1.2 Ca²⁺ channels (the major route for Ca²⁺ entry) open during the action potential plateau, Ca²⁺ flows down a large electrochemical gradient into the cell at rates approaching 10⁶ ions per second. Several pumps and transporters remove Ca²⁺ from the cytoplasm almost as rapidly, returning [Ca²⁺]_i levels back to baseline in time for the next heartbeat. These swift influx and efflux pathways allow the Ca²⁺ signal to achieve its large (tenfold) dynamic range within a single beat and trigger downstream responses within the same time scale.

Translating Changes in Ca²⁺ Into a Cellular Response: CaM as the Prototypical Cardiac Ca²⁺ Sensor

Several Ca²⁺ binding proteins serve as the Ca²⁺ sensors to translate changes in intracellular Ca²⁺ into cellular actions. For example, Ca²⁺ binding to troponin C causes dissociation of the troponin complex from the active site on actin, allowing myosin interaction and force generation. For regulation of ion channels, the best-characterized Ca²⁺ sensor is the ubiquitous Ca²⁺-binding protein CaM, a 16.8 kDa protein that binds four moles of Ca²⁺ per mole of protein. CaM is highly abundant in cardiac myocytes, but more than 98% CaM is apoCaM (Ca²⁺-free) sequestered by binding proteins only to be released upon a significant increase in [Ca²⁺]_i.¹ The affinity of CaM for Ca²⁺ varies significantly depending on whether CaM is free in solution or bound in its apo-state to a target protein, such as an ion channel. Ca²⁺ binding by CaM occurs in the context of 1 to 2 mM intracellular Mg²⁺, the major competing divalent cation. The structural motif in CaM capable of distinguishing Ca²⁺ at levels less than 1 : 1000 of Mg²⁺ is the “EF hand,” a helix-loop-helix domain also found in many other Ca²⁺-binding proteins, including troponin C, in which it was originally identified. CaM has two EF hands in an N-terminal lobular domain and two more in a C-terminal lobular domain. The two Ca²⁺-binding domains are connected by an α-helical segment. Upon Ca²⁺ binding, CaM undergoes significant conformational changes that expose a hydrophobic surface, which can then interact with target proteins in a Ca²⁺-dependent manner. Within most well-characterized target proteins, such as CaMKII, the CaM interaction domain is an amphipathic helix for which the hydrophobic amino acid side chains become buried within the hydrophobic surface exposed in Ca²⁺-saturated CaM. In CaMKII, this amphipathic helix blocks access to the kinase’s constitutively active site; CaM binding to this autoinhibitory domain reveals the active site and thereby endows the kinase with a Ca²⁺-dependent response. In many of the cardiac ion channels to which CaM binds directly, the CaM binding motif has a similar amphipathic pattern. The actions of CaM on the channels, however, are less well understood, and this will be discussed next.

CaM Effector Functions on Channels: Direct Binding and Indirect Actions Through CaMKII

In the last decade there has been an explosion of functional, biochemical, and structural information about how CaM interacts directly with multiple ion channels to affect their function. Details about how CaM regulates the voltage-gated Na⁺ channel Na_V1.5, the L-type voltage-gated Ca²⁺ channel Ca_V1.2, the ryanodine receptor type 2 (RyR2) Ca²⁺ release channel, and several different voltage-gated K⁺ channels, including KCNQ1, will be presented in specific parts of this chapter. The regulatory effects of Ca²⁺ on cardiac ion channels are not mediated solely by CaM interaction with the channels, however, but also indirectly through the actions of other Ca²⁺-regulated proteins, the most prominent of which is CaMKII. In fact, activation of CaMKII results in the phosphorylation of many of the same channels targeted by CaM, and the resulting protein modulation can have profound effects on channel activity, sometimes in ways that oppose the effects of direct CaM interaction with the same channel. Which Ca²⁺-dependent regulator dominates depends on the subcellular location, amplitude, frequency, and duration of the intracellular Ca²⁺ signals, largely because of properties of CaMKII that allow it to target to specific subcellular locations and independently modulate its kinase activity depending on timing of repetitive Ca²⁺ stimuli. Thus, in the heart, CaMKII has the capacity for graded effects that correlate with changes in heart rate.

Several features of CaMKII endow the kinase with this ability to regulate activity depending on the timing of the Ca²⁺ stimuli. These include the holoenzyme’s structure and the fact that CaMKII is itself a CaMKII substrate. The kinase is a homododecamer, in which the 12 subunits are arranged in two stacked hexameric rings with the active site and the CaM-binding autoinhibitory helix of each subunit closest to the periphery. If the Ca²⁺ signal is large enough, or if two Ca²⁺ signals are temporally spaced so that CaM binds simultaneously to two adjacent subunits in a ring, then one of the activated subunits can phosphorylate the other CaM-bound subunit at a specific Thr residue (Thr287 in the cardiac CaMKIIδ isoform) that is adjacent to the CaM binding helix. This phosphorylation event increases the kinase’s affinity for CaM more than 10,000-fold, causing the kinase to become persistently activated even after the activating Ca²⁺ signal has subsided. This capacity to respond differentially to changes in Ca²⁺ influx intensity or frequency make CaMKII therefore ideally suited to respond to changes in cardiac rhythm and the accompanying change in frequency of Ca²⁺ influx through the L-type Ca_V1.2 Ca²⁺ channel with every heartbeat. Thus, CaMKII phosphorylation of the L-type Ca_V1.2 Ca²⁺ channel provides a direct Ca²⁺-dependent means to regulate the major source of Ca²⁺ influx. Finally, because gating of Ca_V1.2 Ca²⁺ channels depends on the concerted actions of Na⁺ and K⁺ channels and the consequent regulation of membrane voltage, CaMKII phosphorylation of any of these channels can provide additional, indirect regulation of Ca_V1.2 Ca²⁺ channels and subsequent Ca²⁺ influx, thereby leading back to CaMKII to generate a web of feedback or feed forward mechanisms to control intracellular Ca²⁺.

Reactive oxygen species provide an additional means to activate CaMKII. Oxidation of two methionine residues, adjacent to the site of transphosphorylation, endows CaMKII with Ca²⁺-independent activity in a manner similar to transphosphorylation.² As with transphosphorylation, Ca²⁺/CaM binding must occur first, exposing the target methionines to oxidation. Given the association of CaMKII with several adverse cardiac outcomes, including arrhythmias, and an increased redox state with those outcomes, this means of activating CaMKII may have important detrimental effects on cardiac ion channels.

Calmodulin Regulation of Cardiac Channel Gating