Many KCNQ1 mutations and rare variants have now been identified in a variety of KCNQ1 domains and regions (http://www.fsm.it/cardmoc/
), some of which are recurrent in populations whilst some remain private to one index case or extended family. This represents a dilemma in the clinical setting because it is difficult to assign pathogenicity to novel variants without several levels of validating evidence, the best of which is the demonstration of biophysical deficits by ‘in-vitro’ electrophysiological experiments. This is a highly-specialised and challenging technology platform which is unlikely to cross over to the diagnostic domain, nevertheless, it can provide definitive evidence and clinical confidence that a gene variant can translate into a functional mutation.
For this reason we describe in this study the biophysical characterisation of 9 KCNQ1 variants in patients with Romano Ward syndrome [15
]. The gene-positive patients had unequivocally prolonged QTc intervals and all but one had typical presentation scenarios such as syncope during exercise, a history of sudden death in the family or resuscitated cardiac arrest. Within the KCNQ1 subunit structure, these mutations are located in different regional domains: the N-terminus (A46T), S5 (T265I), the S5 and P-loop linker (F296S), P-loop (A302V and G316E), S6 (F339S) and the C-terminus (R360G, H445Y, and S546L). Four variants (A46T, A302V, G316E and S546L) have subsequently been detected by other LQTS screening programs, but without any supporting electrophysiological data; whilst there are alternative amino acid substitutions or frameshifts recorded at positions 265 (T265fs+22X), at 302 (A302T), and at 360 (R360T) [10
]. Whatever the status and origin of the KCNQ1 variants, the biophysical investigation of their pathogenicity has not been investigated.
The value of discovering the physiological properties of channel variants has contributed much to the understanding of the pathophysiological mechanisms of LQTS [12
]. Cardiac slowly-activating delayed rectifier K+
) is generated by co-assembly of the α-subunit KCNQ1 (KvLQT1) and its auxiliary subunit KCNE1 (minK). The IKs
current does not play a major role in regulation of cardiac repolarisation under normal physiological conditions. However, mutations in the KCNQ1
gene could cause inherited prolongation of the QT interval by reducing the quality of IKs
current, thereby affecting cardiac repolarisation. Expression studies have demonstrated that LQTS mutations of genes related with K+
currents can produce loss of channel function by either a net current reduction by altering the channel gating and kinetic properties, prevention of assembly of functional channel protein, or an abnormal intracellular protein trafficking.
We examined the electrophysiological properties of the nine KCNQ1 mutations in the presence KCNE1 by heterologous expression in CHO cells and by using a whole-cell voltage clamp. We found seven KCNQ1 mutations causing a reduced IKs current density with co-expression of KCNE1, indicating the loss-of-function in the heterotetrametric state channel. The electrophysiological data for the seven KCNQ1 mutations with decrease the IKs density, suggest several possible mechanisms: (1) a positive shift of the channel activation voltage could lead to a reduction of the current (this effect is more obvious in the mutation S546L); (2) the dominant negative effect, and (3) failure of the channel protein expression within the cell membrane. The studies relating to the latter two effects are separate projects that are being pursued, but irrespective of the specific mechanism involved, it is now possible to label these seven LQTS genotypes as definitive pathological mutations.
Two other mutations (A46T and T265I) displayed no alterations in the current density, although there is evidence that T265I-IKs
displayed a longer delay (~150 msec) before activation, and A46T-IKs
(as well as H445Y-IKs
) lost initial current delay with a rapid activation/rising phase. In the mutation T265I, a longer initial 100-msec current delay and small current magnitude would be responsible for prolonged QT intervals. Studies have demonstrated that the initial delay phase of IKs
channel activation is caused by its β-subunit KCNE1 via moving the channel through multiple closed states before opening during a depolarization [22
], thereby decreasing the initial current activation. In the cases of A46T-IKs and H445Y-IKs, whether the interaction between the mutation and KCNE1 is affected to some degree remains to be studied further. The mutation-caused loss of the delay of the initial IKs
activation could be caused by channel accumulation in open states between depolarization pulses. The mutation caused loss of the delay of the initial IKs activation could be associated with more channel accumulations in Zone 1 of closed states that are near open states in Markove model of the IKs channel kinetics. The channel accumulation in Zone 1 of closed states between cardiac action potentials provides an “available reserve” [24
] which represents an important mechanism for IKs participation in repolarisation and its dependence on rate. During the action potential repolarisation, especially at fast heart rates, these readily available channels can very quickly open on demand to cause rapid IKs activation and rise, an effect to shorten cardiac action potential duration. Again, the current magnitude at the 1st 100-msec of IKs activation was larger in the mutation A46T than in WT current (). Therefore, these alterations in IKs properties could explain failure to observe a prolonged QT interval in the A46T patient.
Structural modelling helps us to conceptualize the different functional effects of particular mutations. Mutation of T265 and F339 affect interactions between S5 and S6. This is consistent with the T265I mutation displaying a longer delay (~150 msec) before activation () as the normal movements of the S5 helix required for efficient opening and closure are impeded by the substitution of threonine by the larger more hydrophobic isoleucine. F339S, on the other hand, involves substitution by a smaller more polar residue, which is less physically impeding, but nevertheless remains pathogenic as indicated by the functional assays ().
Though the A302V substitution involves residues of similar size, the observed functional effects may be due to the external aqueous environment being less forgiving of changes in hydrophobic interactions. Similarly, changes in polarity of exposed extramembrane residues upon mutation affect V0.5
values, resulting accordingly in more negative (F296S, change to polar) or more positive S546L (change to hydrophobic) values. The G316E mutation appears to directly obstruct the normal aperture of the pore by introducing a much larger and negatively charged glutamic acid side-chain to one of the most sensitive gating positions. Modelling and comparison of the heteromeric mutation of two of the four chains () with the wild type () reveals the extent to which the pore is likely to be obstructed. It is interesting to note that the mutation also disrupts the second glycine residue in a combinatorial sequence pattern [S/T]××[S/T]×G[F/Y]G that has been identified in 90% of 134 potassium channel re-entrant loops analysed [25
], which suggests that this glycine residue may also be important for the stable insertion of the P loop within the membrane.
In summary, the biophysical characterisation of these 9 KCNQ1 missense mutations has provided unequivocal heterogeneous proof of pathogenicity in 8 variants to support the various clinical and genetics studies. We remain uncertain as to the pathogenicity of A46T given the suggestive biophysical evidence and co-segregation studies are inconclusive in part due to resistance to wider family screening. In this family it is possible that the genetic basis of the QT prolongation lies elsewhere and further molecular screening of the index cases must remain an option. Whilst cellular electrophysiological testing is unlikely to move easily into the clinical diagnostic area, its clinical value is very important and not in question given the high proportion of novel genetic variants being discovered presenting a challenge to the clinical and genetic counselling teams. For the time-being a collaborative multi-disciplinary approach is needed between clinical and research domains to permit relative and informative proof for cardiologists and genetic counselling teams.