Jeremiah D. Osteen

KCNE1 alters the voltage sensor movements necessary to open the KCNQ1 channel gate

The delayed rectifier IKs potassium channel, formed by coassembly of α- (KCNQ1) and β- (KCNE1) subunits, is essential for cardiac function. Although KCNE1 is necessary to reproduce the functional properties of the native IKs channel, the mechanism(s) through which KCNE1 modulates KCNQ1 is unknown. Here we report measurements of voltage sensor movements in KCNQ1 and KCNQ1/KCNE1 channels using voltage clamp fluorometry. KCNQ1 channels exhibit indistinguishable voltage dependence of fluorescence and current signals, suggesting a one-to-one relationship between voltage sensor movement and channel opening. KCNE1 coexpression dramatically separates the voltage dependence of KCNQ1/KCNE1 current and fluorescence, suggesting an imposed requirement for movements of multiple voltage sensors before KCNQ1/KCNE1 channel opening. This work provides insight into the mechanism by which KCNE1 modulates the IKs channel and presents a mechanism for distinct β-subunit regulation of ion channel proteins.

Allosteric gating mechanism underlies the flexible gating of KCNQ1 potassium channels

KCNQ1 (Kv7.1) is a unique member of the superfamily of voltage-gated K+ channels in that it displays a remarkable range of gating behaviors tuned by coassembly with different β subunits of the KCNE family of proteins. To better understand the basis for the biophysical diversity of KCNQ1 channels, we here investigate the basis of KCNQ1 gating in the absence of β subunits using voltage-clamp fluorometry (VCF). In our previous study, we found the kinetics and voltage dependence of voltage-sensor movements are very similar to those of the channel gate, as if multiple voltage-sensor movements are not required to precede gate opening. Here, we have tested two different hypotheses to explain KCNQ1 gating: (i) KCNQ1 voltage sensors undergo a single concerted movement that leads to channel opening, or (ii) individual voltage-sensor movements lead to channel opening before all voltage sensors have moved. Here, we find that KCNQ1 voltage sensors move relatively independently, but that the channel can conduct before all voltage sensors have activated. We explore a KCNQ1 point mutation that causes some channels to transition to the open state even in the absence of voltage-sensor movement. To interpret these results, we adopt an allosteric gating scheme wherein KCNQ1 is able to transition to the open state after zero to four voltage-sensor movements. This model allows for widely varying gating behavior, depending on the relative strength of the opening transition, and suggests how KCNQ1 could be controlled by coassembly with different KCNE family members.

Biophysical properties of slow potassium channels in human embryonic stem cell derived cardiomyocytes implicate subunit stoichiometry

Non‐technical summaryu2002 The human heart is a pump that works only when its internal electrical system coordinates both its filling and its capacity to eject blood. This critical electrical timing is coordinated by many different ion channels, and this study looks at the one known as IKs. Mutations in its α subunit, KCNQ1, constitute the majority of cases of the disorder long QT syndrome (LQT‐1). Here we have examined properties of human cardiac cells during very early stages of development and found evidence for the manner in which the subunits of IKs assemble; our data suggest that this assembly may be flexible and may change during development and/or disease.

Characterization of KCNQ1 atrial fibrillation mutations reveals distinct dependence on KCNE1.

The IKs potassium channel, critical to control of heart electrical activity, requires assembly of α (KCNQ1) and β (KCNE1) subunits. Inherited mutations in either IKs channel subunit are associated with cardiac arrhythmia syndromes. Two mutations (S140G and V141M) that cause familial atrial fibrillation (AF) are located on adjacent residues in the first membrane-spanning domain of KCNQ1, S1. These mutations impair the deactivation process, causing channels to appear constitutively open. Previous studies suggest that both mutant phenotypes require the presence of KCNE1. Here we found that despite the proximity of these two mutations in the primary protein structure, they display different functional dependence in the presence of KCNE1. In the absence of KCNE1, the S140G mutation, but not V141M, confers a pronounced slowing of channel deactivation and a hyperpolarizing shift in voltage-dependent activation. When coexpressed with KCNE1, both mutants deactivate significantly slower than wild-type KCNQ1/KCNE1 channels. The differential dependence on KCNE1 can be correlated with the physical proximity between these positions and KCNE1 as shown by disulfide cross-linking studies: V141C forms disulfide bonds with cysteine-substituted KCNE1 residues, whereas S140C does not. These results further our understanding of the structural relationship between KCNE1 and KCNQ1 subunits in the IKs channel, and provide mechanisms for understanding the effects on channel deactivation underlying these two atrial fibrillation mutations.

The cardiac IKs channel, complex indeed

The cardiac IKs channel is a major repolarization current in the heart that responds rapidly and robustly to sympathetic nervous system stimulation to ensure adequate diastolic filling time in the face of accompanying accelerated heart rate. In cardiac myocytes, the IKs channel is a macromolecular complex composed of a pore-forming α (KCNQ1) subunit and modulatory β (KCNE1) subunit, as well as intercellular proteins critical for controlling the phosphorylation state of the complex (1). Although KCNQ1 alone assembles to form a voltage-gated potassium channel, the presence of KCNE1 is required to reproduce the kinetic properties of the native IKs channel, and KCNE1 coassembly is necessary to mediate the characteristic IKs response to sympathetic stimulation (2). The importance of the KCNE1 subunit is evidenced by its role in IKs channelopathies: mutation in either KCNQ1 or KCNE1 can underlie congenital long QT syndrome, and the presence of the β subunit is reported to be required for the gain-of-function phenotype in two KCNQ1 mutations implicated in congenital atrial fibrillation (3, 4). Among the questions surrounding the nature of the KCNQ1–KCNE1 association is that of the stoichiometry of the α and β subunits. Because of its homology with the remainder of the Kv channel superfamily, KCNQ1 (Kv7.1) is thought to form channels consisting of four α subunits (5). The number …

Perturbation of sodium channel structure by an inherited Long QT Syndrome mutation

The cardiac voltage-gated sodium channel (NaV1.5) underlies impulse conduction in the heart and its depolarization-induced inactivation is essential in control of the duration of the QT interval of the electrocardiogram (ECG). Perturbation of Nav1.5 inactivation by drugs or inherited mutation can underlie and trigger cardiac arrhythmias. The carboxy terminus plays an important role in channel inactivation, but complete structural information on its predicted structural domain is unknown. Here we measure interactions between the functionally critical distal C-T alpha helix (H6) and the proximal structured EF hand motif using transition metal ion FRET. We measure distances at three loci along H6 relative to an intrinsic tryptophan, demonstrating the proximal-distal interaction in a contiguous carboxy terminus polypeptide. Using these data together with the existing NaV1.5 carboxy terminus NMR structure, we construct a model of the predicted structured region of the carboxy terminus. An arrhythmia associated H6 mutant which impairs inactivation decreases FRET, indicating destabilization of the distal-proximal intramolecular interaction. These data provide a structural correlate to the pathological phenotype of the mutant channel.

Neighboring Alpha-Subunit (KCNQ1) Mutations with a Gain-of-Function IKs Phenotype Show Differential Dependence on Presence of Beta-Subunit (KCNE1)

The IKs cardiac potassium channel forms through co-assembly of KCNQ1, a 6 transmembrane-(TM) spanning voltage-gated potassium channel α subunit and KCNE1, a single TM spanning accessory protein. Two mutations in the S1 transmembrane helix of KCNQ1, S140G and V141M, have been shown to cause a hyperpolarizing shift in the voltage dependence of channel activation and to disrupt deactivation, resulting in accumulation of open channels and a gain-of-function phenotype during repetitive activity that is causally related to congenital human atrial fibrillation. Initial reports suggested that the phenotype of these mutants depends on the presence of the accessory protein KCNE1, which has been shown to be close in proximity to KCNQ1 S1, raising the possibility that KCNE1 directly interacts with KCNQ1 position 140 and/or 141. Here, we show that a Cys substituted at KCNQ1 position 141 spontaneously crosslinks with cysteines introduced in two positions in KCNE1, but a Cys substituted at position 140 does not crosslink to any Cys-substituted KCNE1 residues tested. Co-expression of KCNE1 with either S140G or V141M KCNQ1 slows deactivation and causes similar negative shifts in channel activation. However, in whole-cell patch clamp experiments using isotonic potassium to explore channel deactivation across a wide range of hyperpolarized potentials, we find that the V141M channel activity is indistinguishable from WT while the S140G mutation shifts the V1/2 of activation −30mV and drastically slows deactivation (tau ∼1500ms vs. ∼150ms) when compared with wild-type KCNQ1. Taken together, our results support: 1) an orientation in which KCNQ1 residue V141, but not S140, points toward and is in close proximity to KCNE1 and 2) a direct effect of S140G on channel gating but an allosteric effect of V141M on channel gating that requires the presence of KCNE1.