Formation of Protein Subunits

Following the transcription of RNA from deoxyribonucleic acid (DNA), individual protein monomers are formed via translation on the ribosomes. For proteins destined for the cell membrane, this process occurs only on ribosomes that are attached to the ER, where every three RNA molecules code for an individual amino acid. The generalized pattern is messenger RNA (mRNA) binds to the ribosomes attached to the ER, and transcription proceeds with the nascent polypeptide inserting into the ER membrane. Under normal conditions, protein subunits are folded into their appropriate configuration as the protein is being formed. In some cases, individual monomers interact with other monomers forming channel subunits while still in the ER. These channels leave the ER in membrane-bound vesicles that pinch off as individual packets, sending the individual channels to the Golgi apparatus for post-translational modifications such as glycosylation. From here, channels are delivered to myocyte membranes for insertion and eventual function.

As membrane proteins are formed in the ER, they undergo folding to their correct functional conformation. Occasionally, this process does not work correctly. In order to ensure that nonfunctional proteins are not targeted to the myocyte membrane, a process called unfolded protein response occurs. ER-associated degradation (ERAD) is responsible for the elimination and control of the buildup of aberrant proteins, preventing the aggregation of toxic nonfunctional proteins.¹ These misfolded proteins are recognized by chaperones such as Hsp70/Hsp40, which interact directly with the misfolded protein and aid in its translocation into the cytoplasm for degradation by the proteasome. Evidence is now beginning to accumulate that the ERAD pathway participates not only in the removal of proteins that are not folded correctly but also in the rapid degradation of excess proteins. Thus, a protein is made from RNA in larger quantities than needed under normal conditions but is degraded via the ERAD pathway prior to insertion into cell membranes. When a cell needs a rapid increase of a particular protein, the ERAD system is turned off and more of the protein is trafficked to the cell membrane for use. An example of this is the adenosine-5′-triphosphate (ATP)–sensitive potassium (K[ATP]) channel, whose biogenesis and surface expression are controlled by ERAD. This is thought to be a mechanism for rapid increase in the availability of K(ATP) channels during cellular stress.²

In some cases, actual channel formation from multiple monomers does not occur until after the monomers leave the ER and are within the Golgi apparatus. Here, the monomers form channels (or, in the case of gap junctions, half the channels) and undergo post-translational modifications, which may include glycosylation, acetylation, and interactions with protein partners. In any of these cases, the coordination of this multiple-step process is tightly regulated, and the need for this to work perfectly is underscored by the fact that dysfunction in any of the above steps leads to a loss of ion channel function or gap junction function, altered electrical propagation, and formation of an arrhythmogenic substrate.

Genetic Disorders of Trafficking

Normal trafficking of membrane proteins is exquisitely dependent on having particular amino acid sequences within the protein sequence. These sequences allow for the interaction of the channel protein with molecular motors, cytoskeletal elements, and scaffolding proteins, which are all important for protein movements throughout the cell. Changes in the nucleotide sequence of DNA will translate into RNA missense errors, which, in turn, lead to the production of incorrect amino acid sequences within the proteins. Thus, errors within the genome may produce proteins that are missing part or all of the appropriate trafficking sequences, thus causing genetic trafficking disorders. Since the normal cardiac rhythm is dependent on ion channels and gap junctions, abnormal ion channel and gap junction function underlie a number of cardiac rhythm disturbances. Alteration in individual channel gating function was thought to cause much of the loss of function of mutated channels, but analysis of many of the proarrhythmic mutations indicates that the true cause is the loss of trafficking to the membrane surface. For example, an alteration in the trafficking of ion channels to the cell’s surface has been shown to occur in some forms of atrial fibrillation (AF), long QT syndrome (LQTS) types 1 and 2, Brugada syndrome, and Anderson syndrome (Table 8-1).

One of the more common forms of changes within protein sequences occurs when a coding change produces a protein with alternative amino acids at a given position within the protein. This shift, known as polymorphism, has two possible amino acids at particular loci within a protein but leaves the remaining amino acids in the correct location within the protein sequence. In many cases, no phenotypic change associated with polymorphisms occurs, but in rare cases, the changes at a single site can lead to the production of a protein that is nonfunctional. For example, in SCN5A, a mutation at amino acid 1053 from glutamic acid (E) to lysine (K) leads to a trafficking disorder of the sodium (Na⁺) channel. Written as I1053K, this mutation prevents the SCN5A protein from localizing to cell membranes by interrupting the ability of the SCN5A protein to interact with the scaffolding protein ankyrin G, which normally maintains the protein subunit at the cell membranes (see Chapter 2 and Table 8-1). This simple substitution of a single amino acid thus causes the Brugada syndrome phenotype.

Frame shift mutations may also occur. In these mutations, a coding error causes the initial RNA triplet to have either an extra nucleotide or a lost nucleotide, shifting the reading frame in a manner that causes a novel amino acid to be produced at that site. The produced protein will then have a scrambled or “nonsense” sequence of amino acids. These errors are very severe and often lead to proteins that are trafficking deficient; in addition, if the trafficking deficiency is rescued pharmacologically, these proteins are unable to form a channel with normal function. One example of this is the LQTS type 1 (LQT1) mutation that results from a frame shift at position 178 in the α-subunit of the channel that underlies the slow component of the delayed rectifier current, I_K. In this case, the alanine normally found at position 178 is lost, which leads to the formation of an abnormal amino acid sequence; this sequence ends 105 amino acids later when, by chance, the combination of nucleotides forms a stop codon and the protein translation is terminated. This type of mutation, written as A178fs/105 in this case, causes the formation of a truncated form of the protein that is trafficking deficient and leads to the LQT1 phenotype (see Table 8-1).

Sodium Channels

The primary phenotype found in patients with mutations that cause loss of trafficking of sodium (Na⁺) channels to the cell membranes is Brugada syndrome. This phenotype is associated with high risk for sudden cardiac death. Voltage-gated Na⁺ channels are responsible for the rapid upstroke of the cardiac action potential and persistence of some Na⁺ current after rapid depolarization participates in the early phase of repolarization. Conduction velocity is, in part, dependent on both the amplitude and the rate of activation of these channels, and repolarization is normally slowed by the persistence of Na⁺ inward current during the early plateau phase. Therefore, decreased Na⁺ current leads to a shortened action potential duration. This exaggerates the normal heterogeneity of the outward current found in the heart. This heterogeneity is usually masked by the inward Na⁺ current leading to a voltage gradient across the ventricular wall, which is evident on the ECG as the classic Brugada’s ST-segment elevations in leads V1 through V3.³ Brugada syndrome can also result from mutations in the channel proteins that cause changes in channel function rather than a trafficking defect. Thus, loss of properly functioning Na⁺ channels at the cell membranes—either because of direct Na⁺ channel mutations or through loss of interaction of the Na⁺ channel with trafficking or scaffolding partners—leads to reduced Na⁺ current, increased heterogeneity in repolarization, and the subsequent production of re-entrant arrhythmias leading to sudden cardiac death.

Potassium Channels

Potassium (K⁺) channel mutations that decrease total channel expression at cell membranes are an important cause of LQTS types 1 and 2 in addition to other mutations that cause loss of function of channels that are trafficked to the membranes. The primary current affected in LQTS is the delayed outward rectifier K⁺ current, I_K. Depending on the channel in which a mutation resides, the subtypes of K⁺ current affected (I_Kr or I_Ks) may vary, but the overall effect that manifests as LQTS is a loss of outward K⁺ current. This loss leads to a delay in ventricular cell repolarization and the duration of the QT interval. As with Brugada syndrome, this leads to an increase in transmural repolarization gradients. The mechanism for ventricular tachycardia (torsades de pointes, TdP) associated with LQTS has been postulated to be the occurrence of early after-depolarizations (EADs) and triggered activity caused by the action potential prolongation, which leads to re-entry facilitated by the heterogeneities of repolarization.³

An additional K⁺ channel trafficking mutation associated with cardiac arrhythmias and sudden cardiac death is Andersen-Tawil syndrome (see Table 8-1). This pleiotropic disorder is caused by a primary mutation in the gene KCNJ2. This gene codes for the Kir2.1 channel, which underlies the inwardly rectifying K⁺ channel. As with other K⁺ channel trafficking mutations, the primary ECG manifestation is a longer than normal QT interval, a dispersion of repolarization, and an increase in the propensity for ventricular arrhythmias likely triggered by EADs. While Andersen-Tawil syndrome has electrocardiographic similarities to LQTS, the pleiotropic nature of the disorder (periodic paralysis and dysmorphic features) helps distinguish it from other LQTSs.

Calcium Channels

Unlike Timothy syndrome, which is a gain of function mutation, a trafficking mutation in the calcium (Ca²⁺) channel Cav1.2 is an example of a loss of function mutation leading to a decrease in the L-type Ca²⁺ current. This current normally functions in phase 2 of the action potential causing a sustained depolarization of the cardiac action potential (the plateau phase, mainly ventricular and Purkinje). Thus, loss of this channel leads to loss of the action potential dome, shortening of the action potential, and alterations in the rate of repolarization and duration. As with all cardiac ion channels, the L-type Ca²⁺ channel is heterogeneously expressed with a transmural gradient of expression from the epicardium to the endocardium (high to low current density). Complete channel loss leads to greater dispersion of repolarization across the ventricular wall, manifested as an ST elevation on the ECG, and leads to the formation of the arrhythmogenic substrate. The distinguishing factor between this mutation and other Brugada syndrome mutations is that in patients with this mutation, the QT interval is shorter than normal because of the rapid repolarization of the myocardium.

Gap Junction Channels

Although deficiencies in gap junction function are associated with many different cardiac arrhythmias (see below), to date, the only arrhythmia associated with trafficking deficient mutations of gap junction proteins is AF. In some patients with AF, a subset of trafficking deficient mutations is found in connexin40 (Cx40), a primary gap junction protein in the atria.⁴ Interestingly, these mutations also cause a dominant negative effect on the trafficking of connexin43 (Cx43), the other connexin isoform found in the atria, leading to very low levels of cell–cell coupling between the atrial myocytes. The mechanism for this transdominant effect is not clear, but the overall effect is loss of cell–cell coupling, which leads to slowed conduction and a propensity for the formation of re-entrant arrhythmias. Trafficking mutations in Cx43, which is the major gap junction protein in the ventricular myocardium, have not been found to be associated with ventricular arrhythmias. This is primarily because Cx43 is vital for normal cardiac development and function; thus, trafficking mutations would likely cause embryonic lethality.

Changes in Gap Junction Conductance Without Changes in Connexin Amount or Location

The gap-junctional membrane provides low-resistance pathways for current flow between myocardial cells as well as for the passage of small molecules (up to 1 kDa). The permeability of a gap junction to the flow of ions that carry current (i.e., junctional conductance) is determined by the number of junctional channels, the proportion of the channels that are in the open state, and the permeability (conductance) of the open channel. The permeability of the gap junction channel in the open state, or unitary conductance