Microdomain Interactions of Macromolecular Complexes and Regulation of the Sodium Channel Na V 1.5

Ubiquitin Protein Ligases of the Nedd4/Nedd4-Like Family

Protein ubiquitination tags them for degradation but also serves multiple “nondegradative” functions, in particular membrane protein trafficking. Ubiquitin is a small protein of 76 amino acids found in all animal cells. E3 ubiquitin protein ligases mediate the covalent ubiquitin binding to lysine residues of target proteins. Ubiquitinated membrane proteins are internalized and can be targeted for lysosomal or proteasomal degradation, or deubiquitinated by specific proteases and recycled back to the membrane. ^, The Nedd4-like family of E3 ubiquitin protein ligases regulate many different ion channels. Nedd4-like enzymes specifically bind to target proteins that have consensus domains known as PY motifs with the sequence [L/P]P x Y ( x is any amino acid). ^, Nedd4 and Nedd4-like enzymes harbor several WW domains that can interact with these PY motifs. In Xenopus oocytes, Nedd4-2-mediated ubiquitination decreases I _Na conducted by Na _V 1.5. Our group found that the ubiquitin-protein ligase Nedd4-2 directly binds to the PY motif of Na _V 1.5 and ubiquitinates the channel. Because the total Na _V 1.5 protein level did not decrease upon Nedd4-2 coexpression, we concluded that Nedd4-2 most likely induces increased Na _V 1.5 internalization rather than degradation. On the other hand, inhibition of Nedd4-2 by phosphoinositide 3 kinase may cause increase in Na _V 1.5 expression at the cell membrane. Inhibition of the proteasome is associated with an increase in I _Na and Na _V 1.5 expression in neonatal rat cardiomyocytes. Ubiquitinated Na _V 1.5 was found in mouse cardiac tissue, further suggesting that ubiquitination regulates Na _V membrane turnover or stability in vivo. ^, Interestingly, the effects of Nedd4-2 on Na _V 1.5 partly depend on the intracellular calcium concentration: an increase in intracellular calcium increased Nedd4-2 activity, Nedd4-2-Na _V 1.5 interaction, and Na _V 1.5 ubiquitination in transfected neonatal rat cardiomyocytes and HEK293 cells. Correspondingly, a rat heart failure model showed increased Nedd4-2 and decreased Na _V 1.5 protein levels. Whether these findings correlated with an increase in intracellular calcium was not tested, but cytosolic calcium overload in heart failure is a widely reported phenomenon.

An SCN5A mutation (p.Y1977N) in the Nedd4-2 binding motif constitutes a gain-of-function mutation and is associated with LQTS3. ^, This mutation abolished Na _V 1.5–Nedd4-2 interaction, and prevented Nedd4-2-induced I _Na decrease in HEK293 cells and in cardiomyocytes from p.Y1977N knock-in mice; however, these knock-in mice surprisingly show no changes in action potential parameters or persistent I _Na . On the other hand, an SCN5A mutation introducing an extra PY motif (p.L1239P; from LL x Y to LP x Y) constitutes a loss-of-function of channel function and is associated with BrS. Na _V 1.5-p.L1239P shows increased ubiquitination and degradation in HEK293 cells. Interestingly, the SUMO (small ubiquitin-like modifiers)-conjugating protein UBC9 has been shown to interact with Nedd4-2. UBC9 overexpression increased Na _V 1.5 ubiquitination, reducing Na _V 1.5 protein expression and I _Na . Whether UPC9 only acts on Na _V 1.5 indirectly via Nedd4-2 remains unknown. Altogether, these results strongly suggest that the ubiquitin-proteasome system is involved in Na _V 1.5 regulation and plays a role in cardiac disease.

14-3-3η

The 14-3-3 protein family contains ubiquitous dimeric cytosolic adaptor proteins. The members of this family are involved in many cellular functions, such as the binding and regulation of trafficking of various membrane proteins. Allouis and colleagues performed yeast two-hybrid and coimmunoprecipitation experiments showing that the isoform 14-3-3η interacts with Na _V 1.5 at its N-terminal part of the intracellular loop linking domains I to II (see Fig. 18.1 and Table 18.1 ). 14-3-3 and Na _V 1.5 colocalize at the intercalated discs of rabbit cardiomyocytes. No influence on the Na _V 1.5-mediated peak current was observed when Na _V 1.5 and 14-3-3η were coexpressed in CV-1 in Origin carrying SV40 genetic material (COS) cells, suggesting that this protein does not affect Na _V 1.5 trafficking. In this expression system, 14-3-3 shifted the inactivation curve toward more negative potentials and delayed recovery from inactivation, illustrating that 14-3-3 proteins can modify the biophysical properties of ion channels. Utrilla and coworkers illustrated the complexity of 14-3-3 regulation as inhibition of 14-3-3 proteins did not modify I _Na in Chinese hamster ovary (CHO) cells transfected with Na _V 1.5 only but decreased I _Na and I _K1 when cells were cotransfected with Na _V 1.5 and K _ir 2.1 without abolishing the Na _V 1.5–K _ir 2.1 interaction. Interestingly, 14-3-3 is also proposed to mediate Na _V 1.5 dimerization and coupled gating (i.e., biophysical coupling of two Na _V 1.5 α-subunits). ^{, ,} Because different isoforms of 14-3-3 proteins are expressed in cardiac cells, ^, their exact roles in normal cardiac function and their implications in disease states require further investigation.

Calcium/Calmodulin-Dependent Protein Kinase II

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine protein kinase expressed in many cell types, and it transduces intracellular calcium increases into the phosphorylation of target proteins, including cardiac ion channels. ^, CaMKIIδc is the predominant cardiac isoform upregulated in human and animal heart failure models. ^, Na _V 1.5 colocalizes and coimmunoprecipitates with CaMKIIδc in transfected HEK293 cells, rabbit and rat cardiomyocytes, and mouse heart lysates. ^{, ,} Ashpole and colleagues have shown that CaMKIIδc interacts with the first intracellular loop of Na _V 1.5 (see Fig. 18.1 ) and that residues Ser516 and Thr594 may be phosphorylated by this kinase. Hund and associates found that Ser571 within the same Na _V 1.5 intracellular loop was also a target of CaMKII isoforms α, β, γ, and δ in a β _IV -spectrin-dependent manner. β _IV -spectrin interacts with ankyrin-G at the intercalated discs, suggesting Ser571 phosphorylation may be specific to the intercalated disc. Overexpression of CaMKIIδc in rabbit cardiomyocytes and transgenic mice induced a calcium-dependent hyperpolarizing shift of the steady-state inactivation curve, slowed the recovery from inactivation, and increased the persistent/late I _Na . Similar effects on I _Na are observed with certain congenital LQTS type 3 mutations in SCN5A. In mice, transgenic overexpression of CaMKIIδc leads to chronic heart failure and ventricular tachycardia episodes. In dog ventricular myocytes, increased CaMKII activity increases the persistent I _Na . Whether these observations are the direct consequence of the Na _V 1.5 biophysical alterations or related to other heart failure mechanisms remains unclear. In atrial samples from atrial fibrillation patients, phospho-Na _V 1.5 at Ser571 and autophosphorylated CaMKII were increased, correlating with the increased persistent I _Na and calcium handling defects in murine atria expressing phosphomimetic Na _V 1.5-S571E. Similarly, in atrial samples from patients with sleep-disordered breathing, which is often associated with atrial arrhythmias, activated CaMKII levels, persistent I _Na , and phospho-Na _V 1.5 at Ser571 were increased, whereas peak I _Na was decreased because of a shift in steady-state inactivation. Taken together, CaMKII is an important component in the pathogenesis of arrhythmias and a potential drug target. ^,

Protein Phosphatase 2A

El Rafaey and colleagues recently described protein phosphatase 2A (PP2A) as a negative regulator of the Na _V 1.5/CaMKII axis. PP2A is a serine-threonine phosphatase with a catalytic, scaffolding, and regulatory subunit. Cardiomyocytes from mice lacking the B56α regulatory subunit show increased PP2A activity, less Na _V 1.5 phosphorylation at Ser571, and less persistent sodium current on isoproterenol treatment than wild-type cells. PP2A colocalizes with Na _V 1.5 at the intercalated disc, and interacts with Na _V 1.5, probably mediated by ankyrin-G, as shown in coimmunoprecipitation experiments. Thus PP2A may play an important role in modulating CaMKII-induced Na _V 1.5 phosphorylation and the persistent sodium current.

Protein Tyrosine Phosphatase PTPH1

Ion channels are also regulated by phosphorylation of tyrosine residues, a process that depends both on the phosphorylation activity of tyrosine protein kinases and the dephosphorylation by phosphatases. Overexpression of protein tyrosine kinase Fyn in HEK293 cells was shown to alter several biophysical properties of Na _V 1.5 via the phosphorylation of Tyr1495. This residue is close to the Ile-Phe-Met (IFM) cluster in the intracellular loop linking domains III–IV (see Fig. 18.1 ), which mediates the rapid inactivation of voltage-gated sodium channels. Coexpressing Fyn with Na _V 1.5 in HEK293 cells shifted the I _Na steady-state inactivation curve toward depolarized potentials and accelerated the recovery from inactivation. The interaction site of Fyn with Na _V 1.5 remains to be mapped.

Our group reported that the protein tyrosine phosphatase PTPH1 interacts with the PDZ-domain-binding motif of Na _V 1.5. PTPH1 coexpression in HEK293 cells shifted the availability curve of wild-type Na _V 1.5 toward hyperpolarized potentials. This effect was abolished when the PDZ-domain-binding motif of the Na _V 1.5 C-terminus was removed by an early stop mutation. These results suggest that tyrosine phosphorylation of Na _V 1.5 modulates the stability of the inactivated state. The fact that several proteins (e.g., PTPH1, syntrophin proteins, and SAP97) interact with the same binding domain supports the model that different Na _V 1.5 multiprotein complexes coexist within cardiac cells.