In most ion channels, one protein (e. g., in the case of Na⁺ channels and L‑type Ca²⁺ channels) or a multimer of one or more proteins (e. g., a homotetramer of Kv7.1 proteins in the slow delayed-rectifier K⁺ channel) form the primary pore-forming α subunit through which ions cross the cell membrane. In most cases, the pore-forming α subunit interacts with several regulatory subunits that further regulate ion channel function. For example, interaction between the pore-forming Kv7.1 subunit and KCNE1 β subunits is required to produce the characteristic slow activation kinetics of the slow delayed-rectifier K⁺ current (I_Ks) that contributes to repolarization of the AP (Fig. 2b; [9]). Most other ion channels similarly associate with different β subunits [3, 7]. Interestingly, however, the exact stoichiometry between pore-forming α subunits and regulatory β subunits remains a topic of debate for most ion channels [3, 8]. Besides classical transmembrane β subunits such as KCNE1, a wide range of other interacting proteins, binding to other parts of the pore-forming α subunit, regulate the function of ion channels and Ca²⁺-handling proteins. For example, at least a dozen regulatory subunits have been identified as part of the RyR2 macromolecular complex [1, 10], including FK-506 binding protein 12.6 (FKBP12.6), which enhances cooperation between the four pore-forming α subunits, regulating the stability of the channel’s closed state. In addition, FKBP12.6 plays a role in coupled-gating between different RyR2 channels. This process, whereby two or more connected RyR2 channels can gate simultaneously, is critical for the rapid synchronous initiation of SR Ca²⁺ release and excitation–contraction coupling [10].

The number of ion channels on the membrane of a cardiomyocyte that determine the cardiac AP is dynamically regulated via trafficking, degradation, and recycling pathways [11, 12]. For several ion channels it has been established that the composition of the macromolecular complex plays a major role in their trafficking. For example, for I_Ks, Na⁺ and L‑type Ca²⁺ channels the interaction with regulatory subunits strongly promotes forward trafficking by masking signals present in their α subunits that normally ensure that these subunits get retained in subcellular compartments (so-called endoplasmic reticulum retention signals). Thus, the interaction with regulatory subunits increases the number of functional channels in the plasma membrane [12]. In agreement with this important role in trafficking, transmural differences in the expression of the KChIP2 regulatory protein determine the gradient in transient-outward K⁺ current I_to from epicardial to endocardial layers of the heart [8, 13]. Interestingly, recent evidence has indicated that the composition of the macromolecular complex not only facilitates trafficking, but also determines where exactly these ion channels traffic. Interactions with different components of the actin or microtubule cytoskeleton appear to play a critical role in this targeting [14]. For example, the last three residues of the Na⁺ channel α subunit form a PDZ-binding motif that enables interaction with syntrophin and SAP97 proteins, which is required for expression of the Na⁺ channel macromolecular complex at the lateral membrane, but not at the intercalated disk [15]. Correct targeting of individual macromolecular complexes is necessary for their correct functioning. For example, plasma membrane targeting of Ca²⁺ channel Ca_v1.3 α subunits requires direct interaction with the multifunctional adapter protein ankyrin-B to ensure proper Ca²⁺ channel function [16]. Also, the Ca²⁺-induced Ca²⁺ release from the SR requires a close interaction between RyR2 and L‑type Ca²⁺ channels [1]. This interaction depends in part on junctophilin 2 proteins that interact with RyR2 and the T‑tubular membrane where L‑type Ca²⁺ channels are predominantly located. Likewise, βII spectrin, an actin-associated molecule, is also required for proper RyR2 channel targeting [17]. Thus, the presence of junctophilin 2 and βII spectrin in the RyR2 macromolecular complex is critical for normal excitation–contraction coupling. Finally, interacting proteins may also determine the stability and degradation of ion channels, as has recently been shown for the L‑type Ca²⁺ channel and its interacting protein polycystin 1 [18].

After trafficking to their specific subcellular location, almost all ion channels undergo various posttranslational modifications that dynamically regulate their biophysical properties [19]. Among these posttranslational modifications, phosphorylation by serine/threonine kinases and corresponding dephosphorylation by phosphatases have been most extensively studied [8, 20]. Given the relatively unspecific nature of phosphorylation the individual signaling components need to be highly localized in order to enable specific regulation of individual targets. A‑kinase anchoring proteins (AKAPs) are a family of structurally diverse proteins that bind kinases and phosphatases and anchor them to the macromolecular ion channel complexes [21]. For example, the targeting of protein kinase A (PKA) to the I_Ks macromolecular complex by the AKAP Yotiao is essential for the upregulation of I_Ks in response to β‑adrenoceptor stimulation (Fig. 2b; [9, 22]). Similarly, PKA is targeted to the RyR2 macromolecular complex via mAKAP, although the functional relevance of PKA-dependent RyR2 regulation remains a topic of debate [10, 23]. In addition to kinases and phosphatases, it has been shown that AKAPs also recruit other components of the β‑adrenoceptor signaling cascade to the vicinity of ion channels and Ca²⁺-handling proteins. For example, both Yotiao and mAKAP also bind adenylyl cyclases responsible for cAMP production and phosphodiesterases responsible for cAMP degradation, thereby expanding the fine tuning of local regulation of ion channel function [21, 22]. It is likely that the composition of the macromolecular complex similarly controls other posttranslational modifications. Indeed, caveolin-3 and syntrophin have been suggested to regulate Na⁺ channel S‑nitrosylation [7] and recent data have identified sirtuin 1 deacetylase as part of the Na⁺ channel macromolecular complex controlling acetylation-mediated changes in I_Na [24]. However, in general the molecular mechanisms controlling these other posttranslational modifications are less well established in the heart.

Given the central role of Ca²⁺ in the short- and long-term regulation of cardiac function, it is not surprising that a large number of feedback mechanisms exist that modulate the electrophysiological properties of the heart in response to changes in intracellular Ca²⁺ levels. Calmodulin is a multifunctional Ca²⁺-binding messenger protein expressed ubiquitously in all cells [25]. It is a common constituent of ion channel macromolecular complexes and plays an integral role in the Ca²⁺-dependent regulation of these channels [25]. For example, Ca²⁺ influx through L‑type Ca²⁺ channels undergoes strong autoregulation through Ca²⁺-dependent inactivation of the channel, which critically depends on the interaction of calmodulin with the C‑terminus of the pore-forming α subunit [25]. Similarly, calmodulin binds to RyR2 and stabilizes its gating [26]. Accumulating data indicate that also non-Ca²⁺ channels can undergo Ca²⁺-dependent regulation mediated by calmodulin in their macromolecular complex. These include Na⁺ channels, Ca²⁺-dependent small-conductance K⁺ channels (SK channels) and I_Ks channels, which enable a close bidirectional coupling between cellular electrophysiology and Ca²⁺ handling [3, 8, 25]. Besides calmodulin, other Ca²⁺-binding proteins can regulate the function of different macromolecular complexes. For example, calsequestrin 2 is a Ca²⁺ buffer located in the SR that also modulates gating of RyR2 channels in response to changes in SR Ca²⁺ concentrations [1]. Similarly, SR Ca²⁺ uptake via SERCA2a is negatively regulated by phospholamban and the interaction between SERCA2a and phospholamban is Ca²⁺ dependent [27], providing a way to improve SR Ca²⁺ uptake under conditions of high cytosolic Ca²⁺.

Cardiac electrophysiology is also regulated by mechanical forces acting on cardiomyocytes through a process termed mechano-electric feedback [28]. In addition to mechanically gated channels (e. g., stretch-activated channels), several traditional voltage-gated ion channels, including Na⁺ channels and inward-rectifier K⁺ currents, are now known to be modulated by mechanical forces [28]. For most voltage-gated ion channels the interaction with the lipid bilayer and cytoskeleton plays an essential role in their mechanical modulation [28]. Alternatively, ion channels may be regulated indirectly through mechanosensitive proteins such as polycystin 1, which increases the stability of L‑type Ca²⁺ channels in response to mechanical stretch [16]. As such, the composition of the macromolecular complex may also modulate mechanosensitive regulation. Finally, modulation of the mechanosensitive regulation of these channels by antiarrhythmic drugs, as has been identified for ranolazine and Na⁺ channels [29], may influence their antiarrhythmic efficacy.