Muscle channelopathies.

Muscle channelopathies encompass a wide range of mainly episodic conditions that are characterized by muscle stiffness and weakness. The myotonic conditions, characterized predominantly by stiffness, include myotonia congenita, paramyotonia congenita, and sodium channel myotonia. The periodic paralysis conditions include hypokalemic periodic paralysis, hyperkalemic periodic paralysis, and Andersen-Tawil syndrome. Clinical history is key, and diagnosis is confirmed by next-generation genetic sequencing of a panel of known genes but can also be supplemented by neurophysiology studies and MRI. As genetic testing expands, so have the spectrum of phenotypes seen including pediatric presentations and congenital myopathies. Management of these conditions requires a multidisciplinary approach with extra support needed when patients require anesthetics or when pregnant. Patients with Andersen-Tawil syndrome will also need cardiac input. Diagnosis is important as symptomatic treatment is available for all of these conditions but need to be tailored to the gene and variant of the patient.

Prevalence of genetically confirmed skeletal muscle channelopathies in the era of next generation sequencing.

We provide an up-to-date and accurate minimum point prevalence of genetically defined skeletal muscle channelopathies which is important for understanding the population impact, planning for treatment needs and future clinical trials. Skeletal muscle channelopathies include myotonia congenita (MC), sodium channel myotonia (SCM), paramyotonia congenita (PMC), hyperkalemic periodic paralysis (hyperPP), hypokalemic periodic paralysis (hypoPP) and Andersen- Tawil Syndrome (ATS). Patients referred to the UK national referral centre for skeletal muscle channelopathies and living in UK were included to calculate the minimum point prevalence using the latest data from the Office for National Statistics population estimate. We calculated a minimum point prevalence of all skeletal muscle channelopathies of 1.99/100 000 (95% CI 1.981-1.999). The minimum point prevalence of MC due to CLCN1 variants is 1.13/100 000 (95% CI 1.123-1.137), SCN4A variants which encode for PMC and SCM is 0.35/100 000 (95% CI 0.346 - 0.354) and for periodic paralysis (HyperPP and HypoPP) 0.41/100 000 (95% CI 0.406-0.414). The minimum point prevalence for ATS is 0.1/100 000 (95% CI 0.098-0.102). There has been an overall increase in point prevalence in skeletal muscle channelopathies compared to previous reports, with the biggest increase found to be in MC. This can be attributed to next generation sequencing and advances in clinical, electrophysiological and genetic characterisation of skeletal muscle channelopathies.

Hyperkalemic periodic paralysis associated with a novel missense variant located in the inner pore of Nav1.4.

BACKGROUND: Hyperkalemic periodic paralysis (HyperPP) is an autosomal dominantly inherited disease characterized by episodic paralytic attacks with hyperkalemia, and is caused by mutations of the SCN4A gene encoding the skeletal muscle type voltage-gated sodium channel Nav1.4. The pathological mechanism of HyperPP was suggested to be associated with gain-of-function changes for Nav1.4 gating, some of which are defects of slow inactivation. CASE PRESENTATION & METHODS: We identified a HyperPP family consisting of the proband and his mother, who showed a novel heterozygous SCN4A variant, p.V792G, in an inner pore lesion of segment 6 in Domain II of Nav1.4. Clinical and neurophysiological evaluations were conducted for the proband and his mother. We explored the pathogenesis of the variant by whole-cell patch clamp technique using HEK293T cells expressing the mutant Nav1.4 channel. RESULTS: Functional analysis of Nav1.4 with the V792G mutation revealed a hyperpolarized shift of voltage-dependent activation and fast inactivation. Moreover, steady-state slow inactivation in V792G was impaired with larger residual currents in comparison with wild-type Nav1.4. CONCLUSION: V792G in SCN4A is a pathogenic variant associated with the HyperPP phenotype and the inner pore lesion of Nav1.4 plays a crucial role in slow inactivation.

Periodic paralysis due to cumulative effects of rare variants in SCN4A with small functional alterations.

INTRODUCTION/AIMS: Mutations in the SCN4A gene encoding a voltage-gated sodium channel (Nav1.4) cause hyperkalemic periodic paralysis (HyperPP) and hypokalemic periodic paralysis (HypoPP). Typically, both HyperPP and HypoPP are considered as monogenic disorders caused by a missense mutation with a large functional effect. However, a few cases with atypical periodic paralysis phenotype have been caused by multiple mutations in ion-channel genes expressed in skeletal muscles. In this study we investigated the underlying pathogenic mechanisms in such cases. METHODS: We clinically assessed two families: proband 1 with HyperPP and proband 2 with atypical periodic paralysis with hypokalemia. Genetic analyses were performed by next-generation sequencing and conventional Sanger sequencing, followed by electrophysiological analyses of the mutant Nav1.4 channels expressed in human embryonic kidney 293T (HEK293T) cells using the whole-cell patch-clamp technique. RESULTS: In proband 1, K880del was identified in the SCN4A gene. In proband 2, K880del and a novel mutation, R1639H, were identified in the same allele of the SCN4A gene. Functional analyses revealed that the K880del in SCN4A has a weak functional effect on hNav1.4, increasing the excitability of the sarcolemma, which could represent a potential pathogenic factor. Although R1639H alone did not reveal functional changes strong enough to be pathogenic, Nav1.4 with both K880del and R1639H showed enhanced activation compared with K880del alone, indicating that R1639H may modify the hNav1.4 channel function. DISCUSSION: A cumulative effect of variants with small functional alterations may be considered as the underpinning oligogenic pathogenic mechanisms for the unusual phenotype of periodic paralysis.

Mutation spectrum and health status in skeletal muscle channelopathies in Japan.

Skeletal muscle channelopathies, including non-dystrophic myotonia and periodic paralysis, are rare hereditary disorders caused by mutations of various ion channel genes. To define the frequency of associated mutations of skeletal muscle channelopathies in Japan, clinical and genetic data of two academic institutions, which provides genetic analysis service, were reviewed. Of 105 unrelated pedigrees genetically confirmed, 66 pedigrees were non-dystrophic myotonias [CLCN1 (n = 30) and SCN4A (n = 36)], 11 were hyperkalemic periodic paralysis (SCN4A), and 28 were hypokalemic periodic paralysis [CACNA1S (n = 16) and SCN4A (n = 12)]. Of the 30 families with myotonia congenita, dominant form (Thomsen type) consisted 67%, and unique mutations, A298T, P480T, T539A, and M560T, not found in Western countries, were commonly identified in CLCN1. Hypokalemic periodic paralysis caused by SCN4A mutations consisted 43% in Japan, which was much higher than previous reports. Furthermore, the quality of life of the patients was assessed using the patient-reported outcome measures, SF-36 and INQoL, for 41 patients. This study indicated that the etiology of skeletal muscle channelopathies in Japan was not identical to previous reports from Western countries, and provided crucial information for genetics as well as future therapeutic interventions.

A zebrafish model of nondystrophic myotonia with sodium channelopathy.

Nondystrophic myotonias are disorders of Na+ (Nav1.4 or SCN4A) and Cl- (CLCN1) channels in skeletal muscles, and frequently show phenotype heterogeneity. The molecular mechanism underlying their pathophysiology and phenotype heterogeneity remains unclear. As zebrafish models have been recently exploited for studies of the pathophysiology and phenotype heterogeneity of various human genetic diseases, a zebrafish model may be useful for delineating nondystrophic myotonias. Here, we generated transgenic zebrafish expressing a human mutant allele of SCN4A, referred to as Tg(mylpfa:N440K), and needle electromyography revealed increased number of myotonic discharges and positive sharp waves in the muscles of Tg(mylpfa:N440K) than in controls. In addition, forced exercise test at a water temperature of 24 °C showed a decrease in the distance moved, time spent in and number of visits to the zone with stronger swimming resistance. Finally, a forced exercise test at a water temperature of 18 °C exhibited a higher number of dive-bombing periods and drifting-down behavior than in controls. These findings indicate that Tg(mylpfa:N440K) is a good vertebrate model of exercise- and cold-induced human nondystrophic myotonias. This zebrafish model may contribute to provide insight into the pathophysiology of myotonia in sodium channelopathy and could be used to explore a new therapeutic avenue.

Overlap of periodic paralysis and paramyotonia congenita caused by SCN4A gene mutations two family reports and literature review.

OBJECTIVE: To verify the diagnosis of channelopathies in two families and explore the mechanism of the overlap between periodic paralysis (PP) and paramyotonia congenita (PMC). METHODS: We have studied two cases with overlapping symptoms of episodic weakness and stiffness in our clinical center using a series of assessment including detailed medical history, careful physical examination, laboratory analyses, muscle biopsy, electrophysiological evaluation, and genetic analysis. RESULTS: The first proband and part of his family with the overlap of PMC and hyperkalemic periodic paralysis (HyperPP) has been identified as c.2111C > T (T704M) substitution of the gene SCN4A. The second proband and part of his family with the overlap of PMC and hypokalemic periodic paralysis type 2 (HypoPP2) has been identified as c.4343G > A (R1448H) substitution of the gene SCN4A. In addition, one member of the second family with overlapping symptoms has been identified as a novel mutation c.2111C > T without the mutation c.4343G > A. CONCLUSIONS: SCN4A gene mutations can cause the overlap of PMC and PP (especially the HypoPP2). The clinical symptoms of episodic weakness and stiffness could happen at a different time or temperature. Based on diagnosis assessments such as medical history and muscle biopsy, further evaluations on long-time exercise test, genetic analysis, and patch clamp electrophysiology test need to be done in order to verify the specific subtype of channelopathies. Furthermore, the improvement of one member in the pregnancy period can be used as a reference for the other female in the child-bearing period with T704M.

Lower-extremity magnetic resonance imaging in patients with hyperkalemic periodic paralysis carrying the SCN4A mutation T704M: 30-month follow-up of seven patients.

Hyperkalemic periodic paralysis (hyperKPP) is a muscle channelopathy characterized by recurrent paralytic attacks. Our previous study, in which we conducted whole-body muscle magnetic resonance imaging (MRI) in patients with hyperKPP, revealed muscle atrophy and fatty change in the lower extremity, especially in older persons. The aim of current study was to identify the progression of myopathy in hyperKPP patients had been assessed in the previous study. We performed lower-extremity muscle MRI in seven hyperKPP patients carrying the T704M mutation in the SCN4A gene at an interval of 30 months. Muscle atrophy, edematous change, fatty change, and fat fraction quantified using the Dixon technique were compared with the previous MRI findings. The lower-extremity MRI scan showed progressive muscle pathologic findings when compared with the previous study. Muscle atrophy, edematous change, and fatty change were prominent in the superficial posterior compartment of the lower leg. The follow-up lower-extremity muscle MRI findings provide evidence for chronic progressive myopathy and suggest the usefulness of MRI for assessing disease progression in patients with hyperKPP. This study is meaningful in terms of providing data showing the longitudinal changes of muscles in patients with periodic paralysis.

Spider toxin inhibits gating pore currents underlying periodic paralysis.

Gating pore currents through the voltage-sensing domains (VSDs) of the skeletal muscle voltage-gated sodium channel NaV1.4 underlie hypokalemic periodic paralysis (HypoPP) type 2. Gating modifier toxins target ion channels by modifying the function of the VSDs. We tested the hypothesis that these toxins could function as blockers of the pathogenic gating pore currents. We report that a crab spider toxin Hm-3 from Heriaeus melloteei can inhibit gating pore currents due to mutations affecting the second arginine residue in the S4 helix of VSD-I that we have found in patients with HypoPP and describe here. NMR studies show that Hm-3 partitions into micelles through a hydrophobic cluster formed by aromatic residues and reveal complex formation with VSD-I through electrostatic and hydrophobic interactions with the S3b helix and the S3-S4 extracellular loop. Our data identify VSD-I as a specific binding site for neurotoxins on sodium channels. Gating modifier toxins may constitute useful hits for the treatment of HypoPP.

Spectrum of Nondystrophic Skeletal Muscle Channelopathies in Children.

BACKGROUND: The nondystrophic skeletal muscle channelopathies are a group of disorders caused by mutations of various voltage-gated ion channel genes, including nondystrophic myotonia and periodic paralysis. METHODS: We identified patients with a diagnosis of muscle channelopathy from our neuromuscular database in a tertiary care pediatric center from 2005 to 2015. We then performed a retrospective review of their medical records for demographic characteristics, clinical features, investigations, treatment, and follow-up. RESULTS: Thirty-three patients were identified. Seventeen had nondystrophic myotonia. Seven of them had chloride channelopathy (four Becker disease and three Thomsen disease). Warm-up phenomenon and muscle hypertrophy were common clinical manifestations in this subgroup. Ten patients had sodium channelopathy (four paramyotonia congenita and six other sodium channel myotonia). Stiffness of the facial muscles was an important presenting symptom, and eyelid myotonia was a common clinical finding in this subgroup. The majority of these patients had electrical myotonia. Mexiletine was effective in controlling the symptoms in patients who had received treatment. Sixteen children had periodic paralysis (four hyperkalemic periodic paralysis, eight hypokalemic periodic paralysis, and four Andersen-Tawil syndrome). Acetazolamide was commonly used to prevent paralytic attacks and was found to be effective. CONCLUSIONS: Nondystrophic muscle channelopathies present with diverse clinical manifestations (myotonia, muscle hypertrophy, proximal weakness, swallowing difficulties, and periodic paralysis). Cardiac arrhythmias are potentially life threatening in Andersen-Tawil syndrome. Timely identification of these disorders is helpful for effective symptomatic management and genetic counseling.

A novel Kir2.6 mutation associated with hypokalemic periodic paralysis.

BACKGROUND AND OBJECTIVE: Mutations in KCNJ18, which encodes the inwardly rectifying potassium channel Kir2.6, have rarely been reported in hypokalemic periodic paralysis. We describe the clinical phenotype of a novel KCNJ18 gene mutation and perform functional characterization of this mutant Kir2.6. METHODS: A long-term exercise test (ET) was conducted based on the McManis method. Whole-cell currents were recorded using patch clamp, and the HEK293 cells were transfected with wild-type or/and mutant Kir2.6 cDNA. RESULTS: A de novo conserved heterozygous mutation in Kir2.6, G169R, was found in a hypokalemic periodic paralysis patient. ET led to a decrease in the amplitude of compound muscle action potential (CMAP) by 64%. Patch clamp results showed that the potassium inward and outward current densities of the G169R mutant were, respectively, reduced by 65.6% and 84.7%; for co-expression with wild type, which more closely resembles the physiological conditions in vitro, the inward and outward current densities decreased, respectively, by 48.2% and 47.4%. CONCLUSIONS: A novel KCNJ18 mutation, G169R, was first reported to be associated with hypokalemic periodic paralysis without hyperthyroidism. Electrophysiological results demonstrated a significant functional defect of this mutant, which may predispose patients with this mutation to paralysis. SIGNIFICANCE: This new G169R mutation of the potassium channel Kir2.6 provides insight into the pathogenic mechanisms of hypokalemic periodic paralysis.

Understanding the physiology of the asymptomatic diaphragm of the M1592V hyperkalemic periodic paralysis mouse.

The diaphragm muscle of hyperkalemic periodic paralysis (HyperKPP) patients and of the M1592V HyperKPP mouse model rarely suffers from the myotonic and paralytic symptoms that occur in limb muscles. Enigmatically, HyperKPP diaphragm expresses the mutant NaV1.4 channel and, more importantly, has an abnormally high Na(+) influx similar to that in extensor digitorum longus (EDL) and soleus, two hindlimb muscles suffering from the robust HyperKPP abnormalities. The objective was to uncover the physiological mechanisms that render HyperKPP diaphragm asymptomatic. A first mechanism involves efficient maintenance of resting membrane polarization in HyperKPP diaphragm at various extracellular K(+) concentrations compared with larger membrane depolarizations in HyperKPP EDL and soleus. The improved resting membrane potential (EM) results from significantly increased Na(+) K(+) pump electrogenic activity, and not from an increased protein content. Action potential amplitude was greater in HyperKPP diaphragm than in HyperKPP soleus and EDL, providing a second mechanism for the asymptomatic behavior of the HyperKPP diaphragm. One suggested mechanism for the greater action potential amplitude is lower intracellular Na(+) concentration because of greater Na(+) K(+) pump activity, allowing better Na(+) current during the action potential depolarization phase. Finally, HyperKPP diaphragm had a greater capacity to generate force at depolarized EM compared with wild-type diaphragm. Action potential amplitude was not different between wild-type and HyperKPP diaphragm. There was also no evidence for an increased activity of the Na(+)-Ca(2+) exchanger working in the reverse mode in the HyperKPP diaphragm compared with the wild-type diaphragm. So, a third mechanism remains to be elucidated to fully understand how HyperKPP diaphragm generates more force compared with wild type. Although the mechanism for the greater force at depolarized resting EM remains to be determined, this study provides support for the modulation of the Na(+) K(+) pump as a component of therapy to alleviate weakness in HyperKPP.

¿Cuándo sospechar una parálisis periódica? A propósito de un caso / When to suspect periodic paralysis: report of clinical case

La parálisis periódica es una patología excepcional que afecta a los canales iónicos musculares por diferentes causas. Produce una pérdida de fuerza muscular de manera llamativa y brusca, más evidente en la zona proximal de miembros inferiores. El hallazgo de hipopotasemia coincidiendo con estos ataques nos orienta al diagnóstico y nos muestra su diana terapéutica inicial (AU)

Periodic paralysis (PP) is an unusual disease related to a defect in muscle ion channels and caused by different pathologies. It is characterized by abrupt muscle weakness affecting rather proximal than distal muscles in lower limbs. The finding of hypokalemia during these attacks leads us to a diagnosis of hypokalemic PP and shows its initial therapeutic target (AU)

Contractile abnormalities of mouse muscles expressing hyperkalemic periodic paralysis mutant NaV1.4 channels do not correlate with Na+ influx or channel content.

Hyperkalemic periodic paralysis (HyperKPP) is characterized by myotonic discharges that occur between episodic attacks of paralysis. Individuals with HyperKPP rarely suffer respiratory distress even though diaphragm muscle expresses the same defective Na(+) channel isoform (NaV1.4) that causes symptoms in limb muscles. We tested the hypothesis that the extent of the HyperKPP phenotype (low force generation and shift toward oxidative type I and IIA fibers) in muscle is a function of 1) the NaV1.4 channel content and 2) the Na(+) influx through the defective channels [i.e., the tetrodotoxin (TTX)-sensitive Na(+) influx]. We measured NaV1.4 channel protein content, TTX-sensitive Na(+) influx, force generation, and myosin isoform expression in four muscles from knock-in mice expressing a NaV1.4 isoform corresponding to the human M1592V mutant. The HyperKPP flexor digitorum brevis muscle showed no contractile abnormalities, which correlated well with its low NaV1.4 protein content and by far the lowest TTX-sensitive Na(+) influx. In contrast, diaphragm muscle expressing the HyperKPP mutant contained high levels of NaV1.4 protein and exhibited a TTX-sensitive Na(+) influx that was 22% higher compared with affected extensor digitorum longus (EDL) and soleus muscles. Surprisingly, despite this high burden of Na(+) influx, the contractility phenotype was very mild in mutant diaphragm compared with the robust abnormalities observed in EDL and soleus. This study provides evidence that HyperKPP phenotype does not depend solely on the NaV1.4 content or Na(+) influx and that the diaphragm does not depend solely on Na(+)-K(+) pumps to ameliorate the phenotype.

Characterization of hyperkalemic periodic paralysis: a survey of genetically diagnosed individuals.

This exploratory study aims to create an evidence-based comprehensive characterization of hyperkalemic periodic paralysis (hyperPP). HyperPP is a rare genetic disorder that causes episodes of flaccid paralysis. Disease descriptions in the literature are based upon isolated clinical encounters and case reports. We describe the experience of a large cohort of genetically diagnosed individuals with hyperPP. We surveyed genetically characterized individuals age 18 and over to assess disease comorbidities, diagnostic testing, management, and quality of life issues relevant to hyperPP. Myotonia was reported by 55.8 % of subjects and paramyotonia by 45.3 %. There is a relative risk of 3.6 (p < 0.0001) for thyroid dysfunction compared to the general population. Twenty-five percent of subjects experienced their sentinel attack in the second decade of life. It took an average of 19.4 years and visits to four physicians to arrive at the diagnosis of hyperPP. In addition to limbs and hands being affected during attacks, 26.1 % of subjects reported their breathing musculature was affected and 62.0 % reported their facial muscles were affected. There was a lifelong trend of increasing attack frequency, which was particularly common during childhood and adolescence. Approximately one-third of individuals experienced progressive myopathy. Permanent muscle weakness was evident and worsened during childhood and after age 40. Those with no chronic treatment regimen have a RR of 2.3 for inadequate disease control compared to those taking long-term medications. This study revealed a multitude of heretofore unidentified characteristics of hyperPP, in addition to providing a different perspective on some previously held notions regarding the condition.

Long-term effectiveness of acetazolamide on permanent weakness in hyperkalemic periodic paralysis.

Acetazolamide is commonly used as an empirical treatment for inherited periodic paralyses although some patients may develop deleterious effects. We report a 65 year-old man with hyperkalemic periodic paralysis and late-onset permanent weakness in association with the common T704M mutation in α-subunit, skeletal muscle voltage-gated sodium channel gene. He rapidly recovered from weakness after acetazolamide treatment. Magnetic resonance imaging of thighs comparing pre- and post-treatment revealed a significant increase in muscle bulk. The patient has been without any type of weakness for over 6 years. This data show the remarkable benefit of acetazolamide on permanent weakness of hyperkalemic periodic paralysis in association with the T704M mutation.

Prevalence study of genetically defined skeletal muscle channelopathies in England.

OBJECTIVES: To obtain minimum point prevalence rates for the skeletal muscle channelopathies and to evaluate the frequency distribution of mutations associated with these disorders. METHODS: Analysis of demographic, clinical, electrophysiologic, and genetic data of all patients assessed at our national specialist channelopathy service. Only patients living in the United Kingdom with a genetically defined diagnosis of nondystrophic myotonia or periodic paralysis were eligible for the study. Prevalence rates were estimated for England, December 2011. RESULTS: A total of 665 patients fulfilled the inclusion criteria, of which 593 were living in England, giving a minimum point prevalence of 1.12/100,000 (95% confidence interval [CI] 1.03-1.21). Disease-specific prevalence figures were as follows: myotonia congenita 0.52/100,000 (95% CI 0.46-0.59), paramyotonia congenita 0.17/100,000 (95% CI 0.13-0.20), sodium channel myotonias 0.06/100,000 (95% CI 0.04-0.08), hyperkalemic periodic paralysis 0.17/100,000 (95% CI 0.13-0.20), hypokalemic periodic paralysis 0.13/100,000 (95% CI 0.10-0.17), and Andersen-Tawil syndrome (ATS) 0.08/100,000 (95% CI 0.05-0.10). In the whole sample (665 patients), 15 out of 104 different CLCN1 mutations accounted for 60% of all patients with myotonia congenita, 11 out of 22 SCN4A mutations for 86% of paramyotonia congenita/sodium channel myotonia pedigrees, and 3 out of 17 KCNJ2 mutations for 42% of ATS pedigrees. CONCLUSION: We describe for the first time the overall prevalence of genetically defined skeletal muscle channelopathies in England. Despite the large variety of mutations observed in patients with nondystrophic myotonia and ATS, a limited number accounted for a large proportion of cases.

Voltage-sensor movements describe slow inactivation of voltage-gated sodium channels II: a periodic paralysis mutation in Na(V)1.4 (L689I).

In skeletal muscle, slow inactivation (SI) of Na(V)1.4 voltage-gated sodium channels prevents spontaneous depolarization and fatigue. Inherited mutations in Na(V)1.4 that impair SI disrupt activity-induced regulation of channel availability and predispose patients to hyperkalemic periodic paralysis. In our companion paper in this issue (Silva and Goldstein. 2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210909), the four voltage sensors in Na(V)1.4 responsible for activation of channels over microseconds are shown to slowly immobilize over 1-160 s as SI develops and to regain mobility on recovery from SI. Individual sensor movements assessed via attached fluorescent probes are nonidentical in their voltage dependence, time course, and magnitude: DI and DII track SI onset, and DIII appears to reflect SI recovery. A causal link was inferred by tetrodotoxin (TTX) suppression of both SI onset and immobilization of DI and DII sensors. Here, the association of slow sensor immobilization and SI is verified by study of Na(V)1.4 channels with a hyperkalemic periodic paralysis mutation; L689I produces complex changes in SI, and these are found to manifest directly in altered sensor movements. L689I removes a component of SI with an intermediate time constant (~10 s); the mutation also impedes immobilization of the DI and DII sensors over the same time domain in support of direct mechanistic linkage. A model that recapitulates SI attributes responsibility for intermediate SI to DI and DII (10 s) and a slow component to DIII (100 s), which accounts for residual SI, not impeded by L689I or TTX.