Zoltan Varga

Direct Measurement of Cardiac Na+ Channel Conformations Reveals Molecular Pathologies of Inherited Mutations.

Background—Dysregulation of voltage-gated cardiac Na+ channels (NaV1.5) by inherited mutations, disease-linked remodeling, and drugs causes arrhythmias. The molecular mechanisms whereby the NaV1.5 voltage-sensing domains (VSDs) are perturbed to pathologically or therapeutically modulate Na+ current (INa) have not been specified. Our aim was to correlate INa kinetics with conformational changes within the 4 (DI–DIV) VSDs to define molecular mechanisms of NaV1.5 modulation. Method and Results—Four NaV1.5 constructs were created to track the voltage-dependent kinetics of conformational changes within each VSD, using voltage-clamp fluorometry. Each VSD displayed unique kinetics, consistent with distinct roles in determining INa. In particular, DIII-VSD deactivation kinetics were modulated by depolarizing pulses with durations in the intermediate time domain that modulates late INa. We then used the DII-VSD construct to probe the molecular pathology of 2 Brugada syndrome mutations (A735V and G752R). A735V shifted DII-VSD voltage dependence to depolarized potentials, whereas G752R significantly slowed DII-VSD kinetics. Both mutations slowed INa activation, although DII-VSD activation occurred at higher potentials (A735V) or at later times (G752R) than ionic current activation, indicating that the DII-VSD allosterically regulates the rate of INa activation and myocyte excitability. Conclusions—Our results reveal novel mechanisms whereby the NaV1.5 VSDs regulate channel activation and inactivation. The ability to distinguish distinct molecular mechanisms of proximal Brugada syndrome mutations demonstrates the potential of these methods to reveal how inherited mutations, post-translational modifications, and antiarrhythmic drugs alter NaV1.5 at the molecular level.

Mechanisms of noncovalent β subunit regulation of NaV channel gating

Voltage-gated Na+ (NaV) channels comprise a macromolecular complex whose components tailor channel function. Key components are the non-covalently bound &bgr;1 and &bgr;3 subunits that regulate channel gating, expression, and pharmacology. Here, we probe the molecular basis of this regulation by applying voltage clamp fluorometry to measure how the &bgr; subunits affect the conformational dynamics of the cardiac NaV channel (NaV1.5) voltage-sensing domains (VSDs). The pore-forming NaV1.5 &agr; subunit contains four domains (DI–DIV), each with a VSD. Our results show that &bgr;1 regulates NaV1.5 by modulating the DIV-VSD, whereas &bgr;3 alters channel kinetics mainly through DIII-VSD interaction. Introduction of a quenching tryptophan into the extracellular region of the &bgr;3 transmembrane segment inverted the DIII-VSD fluorescence. Additionally, a fluorophore tethered to &bgr;3 at the same position produced voltage-dependent fluorescence dynamics strongly resembling those of the DIII-VSD. Together, these results provide compelling evidence that &bgr;3 binds proximally to the DIII-VSD. Molecular-level differences in &bgr;1 and &bgr;3 interaction with the &agr; subunit lead to distinct activation and inactivation recovery kinetics, significantly affecting NaV channel regulation of cell excitability.

Molecular motions that shape the cardiac action potential: Insights from voltage clamp fluorometry

Very recently, voltage-clamp fluorometry (VCF) protocols have been developed to observe the membrane proteins responsible for carrying the ventricular ionic currents that form the action potential (AP), including those carried by the cardiac Na(+) channel, NaV1.5, the L-type Ca(2+) channel, CaV1.2, the Na(+)/K(+) ATPase, and the rapid and slow components of the delayed rectifier, KV11.1 and KV7.1. This development is significant, because VCF enables simultaneous observation of ionic current kinetics with conformational changes occurring within specific channel domains. The ability gained from VCF, to connect nanoscale molecular movement to ion channel function has revealed how the voltage-sensing domains (VSDs) control ion flux through channel pores, mechanisms of post-translational regulation and the molecular pathology of inherited mutations. In the future, we expect that this data will be of great use for the creation of multi-scale computational AP models that explicitly represent ion channel conformations, connecting molecular, cell and tissue electrophysiology. Here, we review the VCF protocol, recent results, and discuss potential future developments, including potential use of these experimental findings to create novel computational models.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu. Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability. The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques. In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest(1). This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

Regulation of Na+ channel inactivation by the DIII and DIV voltage-sensing domains

Functional eukaryotic voltage-gated Na+ (NaV) channels comprise four domains (DI–DIV), each containing six membrane-spanning segments (S1–S6). Voltage sensing is accomplished by the first four membrane-spanning segments (S1–S4), which together form a voltage-sensing domain (VSD). A critical NaV channel gating process, inactivation, has previously been linked to activation of the VSDs in DIII and DIV. Here, we probe this interaction by using voltage-clamp fluorometry to observe VSD kinetics in the presence of mutations at locations that have been shown to impair NaV channel inactivation. These locations include the DIII–DIV linker, the DIII S4–S5 linker, and the DIV S4-S5 linker. Our results show that, within the 10-ms timeframe of fast inactivation, the DIV-VSD is the primary regulator of inactivation. However, after longer 100-ms pulses, the DIII–DIV linker slows DIII-VSD deactivation, and the rate of DIII deactivation correlates strongly with the rate of recovery from inactivation. Our results imply that, over the course of an action potential, DIV-VSDs regulate the onset of fast inactivation while DIII-VSDs determine its recovery.

An engineered scorpion toxin analogue with improved Kv1.3 selectivity displays reduced conformational flexibility

The voltage-gated Kv1.3 K+ channel plays a key role in the activation of T lymphocytes. Kv1.3 blockers selectively suppress immune responses mediated by effector memory T cells, which indicates the great potential of selective Kv1.3 inhibitors in the therapy of certain autoimmune diseases. Anuroctoxin (AnTx), a 35-amino-acid scorpion toxin is a high affinity blocker of Kv1.3, but also blocks Kv1.2 with similar potency. We designed and produced three AnTx variants: ([F32T]-AnTx, [N17A]-AnTx, [N17A/F32T]-AnTx) using solid-phase synthesis with the goal of improving the selectivity of the toxin for Kv1.3 over Kv1.2 while keeping the high affinity for Kv1.3. We used the patch-clamp technique to determine the blocking potency of the synthetic toxins on hKv1.3, mKv1.1, hKv1.2 and hKCa3.1 channels. Of the three variants [N17A/F32T]-AnTx maintained the high affinity of the natural peptide for Kv1.3 but became more than 16000-fold selective over Kv1.2. NMR data and molecular dynamics simulations suggest that the more rigid structure with restricted conformational space of the double substituted toxin compared to the flexible wild-type one is an important determinant of toxin selectivity. Our results provide the foundation for the possibility of the production and future therapeutic application of additional, even more selective toxins targeting various ion channels.

7DHC-induced changes of Kv1.3 operation contributes to modified T cell function in Smith-Lemli-Opitz syndrome

In vitro manipulation of membrane sterol level affects the regulation of ion channels and consequently certain cellular functions; however, a comprehensive study that confirms the pathophysiological significance of these results is missing. The malfunction of 7-dehydrocholesterol (7DHC) reductase in Smith-Lemli-Opitz syndrome (SLOS) leads to the elevation of the 7-dehydrocholesterol level in the plasma membrane. T lymphocytes were isolated from SLOS patients to assess the effect of the in vivo altered membrane sterol composition on the operation of the voltage-gated Kv1.3 channel and the ion channel-dependent mitogenic responses. We found that the kinetic and equilibrium parameters of Kv1.3 activation changed in SLOS cells. Identical changes in Kv1.3 operation were observed when control/healthy T cells were loaded with 7DHC. Removal of the putative sterol binding sites on Kv1.3 resulted in a phenotype that was not influenced by the elevation in membrane sterol level. Functional assays exhibited impaired activation and proliferation rate of T cells probably partially due to the modified Kv1.3 operation. We concluded that the altered membrane sterol composition hindered the operation of Kv1.3 as well as the ion channel-controlled T cell functions.

Optimization of the Synthesis of Flavone-Amino Acid and Flavone-Dipeptide Hybrids via Buchwald-Hartwig Reaction

The article describes the development of Buchwald-Hartwig amination of different bromoflavones with amino acid and peptide derivatives as nitrogen source giving unique structures. The previously observed racemization, which occurred during the synthesis of flavone-amino acid hybrids, was successfully prevented in most cases. The biological assays of these novel structures showed cytotoxic effects on different cancer cell lines.

Impact of Consultation on Hospital Outcomes and Resource Utilization for Patients with Acute Congestive Heart Failure

Objectives Consultation is an important tool for acquiring subspecialty support when managing patients with acute congestive heart failure (CHF). The effect of consultation on hospital outcomes and resource utilization in CHF is unknown. The objectives of our study were to determine the effect of consultation on outcomes in CHF and to evaluate factors affecting the frequency of consultation. Methods Our study was a retrospective cohort study of patients admitted to Florida Hospital Orlando for CHF between January 1, 2011 and December 31, 2013. Data on demographics, number of consultations, length of stay (LOS), readmissions within 30 days, cost of care, and mortality were compared according to the number of consultations. For statistical analysis, analysis of variance, the &khgr;2 test, and multivariate linear regression analysis were used. Risk-adjusted outcomes were reported as observed/expected. Results A total of 1554 patients were included; 103 (6.6%) patients received no consultation; 482 (31%) received 1; 365 (23.5%) received 2; 229 (14%) received 3; and 375 (24%) received ≥4. Teaching service, age, and African American race were associated with decreased consultation (P < 0.001 for all) and high case-mix index was associated with increased consultation (P < 0.001). Adjusted LOS and costs increased with an increased number of consultations (P < 0.001 for both). There was no difference in adjusted mortality or 30-day readmission rate based on the number of consultations (P = 0.35 and 0.98, respectively). Conclusions Increased consultation with patients with CHF is associated with increased costs and LOS without improved mortality or readmission rate. Decreased utilization of consultations by the teaching service suggests that there is an opportunity to decrease utilization of healthcare resources by streamlining the utilization of consultations.

Sterol Regulation of Voltage-Gated K+ Channels

Cholesterol is an essential lipid building block of the cellular plasma membrane. In addition to its structural role, it regulates the fluidity and raft structure of the membrane and influences the course of numerous membrane-linked signaling pathways and the function of transmembrane proteins, including ion channels. This is supported by a vast body of scientific data, which demonstrates the modulation of ion channels with a great variety of ion selectivity, gating, and tissue distribution by changes in membrane cholesterol. Here, we review what is currently known about the modulation of voltage-gated K+ (Kv) channels by changes in membrane cholesterol content, considering raft association of the channels, the roles of cholesterol recognition sites, and those of adaptor proteins in cholesterol-Kv channel interactions. We specifically focus on Kv1.3, the dominant K+ channel of human T cells. Effects of cholesterol depletion and enrichment and 7-dehydrocholesterol enrichment on Kv1.3 gating are discussed in the context of the immunological synapse and the comparison of the in vitro effects of sterol modifications on Kv1.3 function with ex vivo effects on cells from hypercholesterolemic and Smith-Lemli-Opitz patients.