Can robots patch-clamp as well as humans? Characterization of a novel sodium channel mutation

Department of Neurology, Center for Neuroscience and Regeneration Research, Yale University School of Medicine, New Haven, CT 06510, USA.
The Journal of Physiology (Impact Factor: 5.04). 06/2010; 588(Pt 11):1915-27. DOI: 10.1113/jphysiol.2009.186114
Source: PubMed


Ion channel missense mutations cause disorders of excitability by changing channel biophysical properties. As an increasing number of new naturally occurring mutations have been identified, and the number of other mutations produced by molecular approaches such as in situ mutagenesis has increased, the need for functional analysis by patch-clamp has become rate limiting. Here we compare a patch-clamp robot using planar-chip technology with human patch-clamp in a functional assessment of a previously undescribed Nav1.7 sodium channel mutation, S211P, which causes erythromelalgia. This robotic patch-clamp device can increase throughput (the number of cells analysed per day) by 3- to 10-fold. Both modes of analysis show that the mutation hyperpolarizes activation voltage dependence (8 mV by manual profiling, 11 mV by robotic profiling), alters steady-state fast inactivation so that it requires an additional Boltzmann function for a second fraction of total current (approximately 20% manual, approximately 40% robotic), and enhances slow inactivation (hyperpolarizing shift--15 mV by human,--13 mV robotic). Manual patch-clamping demonstrated slower deactivation and enhanced (approximately 2-fold) ramp response for the mutant channel while robotic recording did not, possibly due to increased temperature and reduced signal-to-noise ratio on the robotic platform. If robotic profiling is used to screen ion channel mutations, we recommend that each measurement or protocol be validated by initial comparison to manual recording. With this caveat, we suggest that, if results are interpreted cautiously, robotic patch-clamp can be used with supervision and subsequent confirmation from human physiologists to facilitate the initial profiling of a variety of electrophysiological parameters of ion channel mutations.

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Available from: Mark Estacion
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    • "Thus far, 17 mutations (as shown in Fig. 1C, except for N395K, Q875E, and novel mutation, V1316A) relating to PE have been characterized and all mutant Nav1.7 channels exhibit significant hyperpolarizing shift in voltage dependent activation compared with wild type channel [19], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37]. The mutant channels also produce depolarizing shift in steady-state fast inactivation [23], [24], [32], [34], [35], [37], slow inactivation [19], [25], [26], [28], [30], [31], [32], [33], [36], [37], slow deactivation [19], [24], [25], [26], [28], [29], [30], [31], [32], [34], [35], [36], and increased ramp current [19], [24], [25], [26], [27], [28], [29], [30], [31], [32], [34], [35], [36]. These channel property changes may confer to the hyperexcitibility in DRG neurons. "
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    ABSTRACT: Primary erythromelalgia (PE) is an autosomal dominant neurological disorder characterized by severe burning pain and erythema in the extremities upon heat stimuli or exercise. Mutations in human SCN9A gene, encoding the α-subunit of the voltage-gated sodium channel, Na(v)1.7, were found to be responsible for PE. Three missense mutations of SCN9A gene have recently been identified in Taiwanese patients including a familial (I136V) and two sporadic mutations (I848T, V1316A). V1316A is a novel mutation and has not been characterized yet. Topologically, I136V is located in DI/S1 segment and both I848T and V1316A are located in S4-S5 linker region of DII and DIII domains, respectively. To characterize the elelctrophysiological manifestations, the channel conductance with whole-cell patch clamp was recorded on the over-expressed Chinese hamster overy cells. As compared with wild type, the mutant channels showed a significant hyperpolarizing shift in voltage dependent activation and a depolarizing shift in steady-state fast inactivation. The recovery time from channel inactivation is faster in the mutant than in the wild type channels. Since warmth can trigger and exacerbate symptoms, we then examine the influence of tempearture on the sodium channel conduction. At 35°C, I136V and V1316A mutant channels exhibit a further hyperpolarizing shift at activation as compared with wild type channel, even though wild type channel also produced a significant hyperpolarizing shift compared to that of 25°C. High temperature caused a significant depolarizing shift in steady-state fast inactivation in all three mutant channels. These findings may confer to the hyperexcitability of sensory neurons, especially at high temperature. In order to identifying an effective treatment, we tested the IC(50) values of selective sodium channel blockers, lidocaine and mexiletine. The IC(50) for mexiletine is lower for I848T mutant channel as compared to that of the wild type and other two mutants which is comparable to the clinical observations.
    Full-text · Article · Jan 2013 · PLoS ONE
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    • "With a preconditioning pulse of 10 s, no complete slow inactivation was achieved; therefore, resulting values may not compare to previously published data for slow inactivation in WT Nav1.7. However, a time window of 10 s is considered to be relevant for inducing slow inactivation (Estacion et al. 2010), and therefore, differences seen in mutated Nav1.7 channels compared to WT are representative of enhanced slow inactivation. To investigate the time to recovery from fast inactivation, first a 20-ms pulse to -10 mV was applied followed by a recovery phase between 5 and 100 ms and a second pulse to -10 mV (Cummins et al. 2009). "
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    ABSTRACT: We identified and clinically investigated two patients with primary erythromelalgia mutations (PEM), which are the first reported to map to the fourth domain of Nav1.7 (DIV). The identified mutations (A1746G and W1538R) were cloned and transfected to cell cultures followed by electrophysiological analysis in whole-cell configuration. The investigated patients presented with PEM, while age of onset was very different (3 vs. 61 years of age). Electrophysiological characterization revealed that the early onset A1746G mutation leads to a marked hyperpolarizing shift in voltage dependence of steady-state activation, larger window currents, faster activation kinetics (time-to-peak current) and recovery from steady-state inactivation compared to wild-type Nav1.7, indicating a pronounced gain-of-function. Furthermore, we found a hyperpolarizing shift in voltage dependence of slow inactivation, which is another feature commonly found in Nav1.7 mutations associated with PEM. In silico neuron simulation revealed reduced firing thresholds and increased repetitive firing, both indicating hyperexcitability. The late-onset W1538R mutation also revealed gain-of-function properties, although to a lesser extent. Our findings demonstrate that mutations encoding for DIV of Nav1.7 can not only be linked to congenital insensitivity to pain or paroxysmal extreme pain disorder but can also be causative of PEM, if voltage dependency of channel activation is affected. This supports the view that the degree of biophysical property changes caused by a mutation may have an impact on age of clinical manifestation of PEM. In summary, these findings extent the genotype–phenotype correlation profile for SCN9A and highlight a new region of Nav1.7 that is implicated in PEM. Electronic supplementary material The online version of this article (doi:10.1007/s12017-012-8216-8) contains supplementary material, which is available to authorized users.
    Full-text · Article · Jan 2013 · Neuromolecular medicine
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    • "Single cell and patch approaches provide an experimental template for examination of potential pharmaceutical interventions, but these are notoriously low throughput. Recently, multipatch platforms have been developed to extend cellular electrophysiology studies into the high-throughput realm [Estacion et al. 2010]. Basic voltage-gated ion channel structure Voltage-gated ion channels gate predominantly in response to voltage. "
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    ABSTRACT: Gastrointestinal (GI) functional and motility disorders are highly prevalent and responsible for long-term morbidity and sometimes mortality in the affected patients. It is estimated that one in three persons has a GI functional or motility disorder. However, diagnosis and treatment of these widespread conditions remains challenging. This partly stems from the multisystem pathophysiology, including processing abnormalities in the central and peripheral (enteric) nervous systems and motor dysfunction in the GI wall. Interstitial cells of Cajal (ICCs) are central to the generation and propagation of the cyclical electrical activity and smooth muscle cells (SMCs) are responsible for electromechanical coupling. In these and other excitable cells voltage-sensitive ion channels (VSICs) are the main molecular units that generate and regulate electrical activity. Thus, VSICs are potential targets for intervention in GI motility disorders. Research in this area has flourished with advances in the experimental methods in molecular and structural biology and electrophysiology. However, our understanding of the molecular mechanisms responsible for the complex and variable electrical behavior of ICCs and SMCs remains incomplete. In this review, we focus on the slow waves and action potentials in ICCs and SMCs. We describe the constituent VSICs, which include voltage-gated sodium (Na(V)), calcium (Ca(V)), potassium (K(V), K(Ca)), chloride (Cl(-)) and nonselective ion channels (transient receptor potentials [TRPs]). VSICs have significant structural homology and common functional mechanisms. We outline the approaches and limitations and provide examples of targeting VSICs at the pores, voltage sensors and alternatively spliced sites. Rational drug design can come from an integrated view of the structure and mechanisms of gating and activation by voltage or mechanical stress.
    Full-text · Article · Jan 2012 · Therapeutic Advances in Gastroenterology
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