Sympathetic stimulation also leads to a PKA-dependent increase in a slowly activating potassium channel current, I_Ks (Figure 11-1). I_Ks channels consist of the pore-forming α-subunit KCNQ1 and the auxiliary β-subunit KCNE1^3,4 and contribute to cardiac repolarization. PKA-dependent modulation increases the repolarization current to counter the stimulatory effects of PKA on L-type calcium channels⁵ to achieve a balance of inward and outward membrane currents. This balance of modulated currents is thought of as a necessary mechanism to regulate calcium homeostasis in the face of sympathetic activity.

Figure 11-1 SNS regulation of I_Ks currents. Activation of the β-adrenergic receptor (β-AR) by norepinephrine (NE) results in an increase in the intracellular cAMP concentration, which, in turn, turns on PKA. I_Ks channels are phosphorylated by PKA. This regulation is mediated by a macromolecular complex coordinated by AKAP9. SNS, Sympathetic nervous system; NE, norepinephrine; β-AR, β-adrenergic receptor; PKA, protein kinase A; AKAP, A-kinase anchoring protein; ICaL, L-type Calcium channel.

Inherited mutations in ion channels have been associated with disorders that are exacerbated by SNS activity. For example, the genes that encode KCNQ1 and KCNE1 3,4 have been linked to the congenital long QT syndrome (LQTS). LQTS, a rare disease in which the QT interval of the ECG is prolonged as the result of dysfunctional ventricular repolarization, can precipitate lethal polymorphic ventricular tachycardia and is associated with syncope, seizures, and sudden death.⁶ Mutations in KCNQ1 cause LQT-1, and mutations in KCNE1 cause LQT-5.⁷ In affected patients, triggers of arrhythmias are gene-specific, and those with mutations in KCNQ1 or KCNE1 are at greatest risk of experiencing fatal cardiac arrhythmias in the face of elevated SNS activity.⁸ Unraveling the molecular links between the SNS and regulation of the KCNQ1/KCNE1 channel has direct implications for the mechanistic basis of triggers of arrhythmias in LQTS.

β-AR Signaling: Coordination of Localized Regulation of Channel Proteins by A-Kinase Anchoring Proteins

Key to the complex regulation of ion channels by β-AR stimulation is the spatiotemporal control of local cAMP concentration. This is mediated by the A-kinase anchoring proteins (AKAPs). AKAPs are a group of structurally diverse proteins with the common function of binding to, but not being limited to, PKA regulatory subunits.9–16 AKAPs provide structural scaffolding to integrate various enzymes and their substrates to form a compartmentalized environment. This enables spatiotemporal control of the regulatory enzymes (i.e., to present the enzymes at high concentrations at the site of their substrates when needed).¹⁷ Disruption of local complexes can unbalance the response and may have pathophysiological consequences.

The I_Ks channel forms a macromolecular signaling complex that is coordinated by the binding of AKAP9, also known as yotiao,18, 19 via a leucine zipper (LZ) motif in the KCNQ1 carboxy (C)-terminus domain. Recent studies suggest that AKAP9 associates not only with the PKA regulatory subunit (RII), but also with protein phosphatase 1 (PP1),¹⁸ phosphodiesterase (PDE),²⁰ and adenylyl cyclase (AC)^21–23 (Figure 11-2). Together these enzymes control the phosphorylation state of the channel via the cAMP/PKA pathway.

Figure 11-2 The KCNQ1/KCNE1/AKAP9 macromolecular complex. AKAP9 recruits PKA, phosphatase 1 (PP1), adenylyl cyclase 9 (AC9), and phosphodiesterase 4D3 (PDE4D3) to regulate the phosphorylation state of I_Ks channels.

Role of Leucine Zipper in Protein-Protein Interaction

Marks and colleagues were the first to show that the cardiac calcium release channel/RyR2 is regulated by a macromolecular signaling complex in which kinases and phosphatases are targeted to the channel via AKAP and leucine/isoleucine zipper (LZ) motifs.2,24,25 They suggested that this may be a common motif for coordination of ion channel signaling complexes.²⁵ Subsequent investigations have shown that this is, in fact, the case for at least two other ion channels that are regulated by PKA: L-type calcium channels^{26, 27} and KCNQ1/KCNE1 potassium channels.¹⁸ The LZ domain is an α-helical structure that forms coiled coils and was originally identified as highly conserved motifs mediating the binding of transcription factors to DNA.²⁸ Coiled coils are composed of heptad repeats (abcdefg)_n in which hydrophobic residues occur at positions “a” and “d” and form the hydrophobic face of the helix, while “b, c, e, f, and g” are hydrophilic residues that form the solvent-exposed part of the coiled coil.²⁹ LZs classically contain a leucine in position “d” because of its flexible side chain, although the canonical leucine residue can be replaced by an isoleucine or valine. Electrostatic interactions between side chains in the “e” and “g” sites from neighboring helices are believed to help specify binding partners.³⁰ In vitro site-directed mutagenesis studies have successfully revealed the sequence specificity of interacting helices in proteins such as the GCN4 DNA binding domain,³¹ phospholamban,³² the myosin binding subunit/cGKIα,³³ and ryanodine receptor types 1 and 2.²⁵ In the case of the I_Ks channel (KCNQ1/KCNE1), Marx et al. identified an LZ motif in the C-terminus of the KCNQ1 subunit critical to AKAP9 interaction.¹⁸ Substitution of an alanine for one or more of the “d” position leucines or isoleucines in the LZ motif disintegrated the I_Ks/AKAP9 complex and rendered the channel unable to be regulated by PKA.

Molecular Components of the I_ks/AKAP9 Complex

Associated with the I_Ks/AKAP9 complex are two pairs of enzymes with opposing effects on channel phosphorylation state. The first pair, which involves PKA and PP1, phosphorylates or de-phosphorylates the channel, respectively. The second pair involves AC and PDE, which increase and decrease the local cAMP gradient, respectively (see Figure 11-2). Working in concert, these enzymes fine-tune the I_Ks channel function.³⁴

• PKA. AKAP9-bound PKA regulates I_Ks channel function by phosphorylation of a single amino acid residue (Ser27) on the N-terminus of KCNQ1.¹⁸ The biophysical consequence of PKA phosphorylation of the I_Ks channel is a profound increase in current amplitude, an accelerated onset of activation, a hyperpolarizing shift in the voltage dependence of channel activation, and a slowing of deactivation (the return of activated [open] to resting [closed] channels during diastole).³⁵ The combined effect ensures that during voltage depolarization, KCNQ1/KCNE1 channel activity is increased in the presence of SNS stimulation. Consequently, the substantial repolarization reserve is activated in the face of SNS-mediated activity, and this reserve potassium channel current can offset SNS-mediated increases in calcium channel currents, which would prolong APD.⁵ It is interesting to note that AKAP9 itself was shown to be a substrate of PKA. Indeed, the phosphorylation of AKAP9 seemed to participate in the regulation of I_Ks channels.³⁶ Early works identified a region on AKAP9 (residues 1440-1457: LEEEVAKVIVSMSIAFAQ) as the primary binding site for PKA RII subunits.^37,38

• PP1. PP1 is a nonspecific serine/threonine phosphatase that dephosphorylates its substrate. AKAP9 provides a platform that allows PP1 to specifically target the I_Ks channel.¹⁸ Thus, PP1 can reverse the effect of PKA on the channel and attenuate the channel activity. This was evidenced by an experiment in which it was found that addition of okadaic acid, a PP1 inhibitor, enhanced the effect of PKA-dependent I_Ks channel regulation.¹⁸ The PP1 binding site on AKAP9 was shown to be located in a region that comprises residues 1171-1229.³⁷

• AC. Upstream in the PKA pathway, ACs are activated by Gs-coupled receptors, such as the β-ARs. ACs are responsible for cAMP synthesis, which then activates PKA. Evidence now suggests that ACs are associated with various AKAPs, such as AKAP79/150, mAKAP, and AKAP9.21,39–41 AKAP9 associates with AC1, 2, 3, and 9 but not 4, 5, and 6. Residues 808-957 of AKAP9 bind directly to the AC2 N-terminus. Expression of AKAP9 inhibited the activity of AC2 and 3, but not AC1 or 9.²² It has been demonstrated that AC9 was a member of the I_Ks/AKAP9 complex in the cardiac myocytes of both I_Ks transgenic mice and guinea pigs. AC9 association with the complex sensitizes PKA phosphorylation of KCNQ1 to SNS stimulation.²³

• PDE. Phosphodiesterases (PDEs) constitute the sole route for degrading cyclic nucleotides in cells. In the mammalian heart, the temporal and spatial dynamics of cAMP gradients are controlled mainly by PDE3s and PDE4s with a prevailing role of PDE4s, which are considered the cAMP-specific PDEs.42–44 In transgenic murine cardiac myocytes expressing I_Ks channels, PDE4s were shown to regulate the basal phosphorylation level of I_Ks channels. Two PDE4D isoforms, likely PDE4D3 and PDE4D5, were found to interact with the channels.²⁰ However, in the heterologous expression system, only PDE4D3, which is known to interact with AKAPs (AKAP-18,⁴⁵ AKAP-250,^46,47 and AKAP-450^47,48), was shown to be specifically recruited to KCNQ1 by AKAP9 and to regulate the amplitude of channels in response to cAMP stimulation.²⁰ The binding site for PDE4D3 on AKAP9 currently is not known.