Two small-molecule activators share similar effector sites in the KCNQ1 channel pore but have distinct effects on voltage sensor movements.

ML277 and R-L3 are two small-molecule activators of KCNQ1, the pore-forming subunit of the slowly activating potassium channel IKs. KCNQ1 loss-of-function mutations prolong cardiac action potential duration and are associated with long QT syndrome, which predispose patients to lethal ventricular arrhythmia. ML277 and R-L3 enhance KCNQ1 current amplitude and slow deactivation. However, the presence of KCNE1, an auxiliary subunit of IKs channels, renders the channel insensitive to both activators. We found that ML277 effects are dependent on several residues in the KCNQ1 pore domain. Some of these residues are also necessary for R-L3 effects. These residues form a putative hydrophobic pocket located between two adjacent KCNQ1 subunits, where KCNE1 subunits are thought to dwell, thus providing an explanation for how KCNE1 renders the IKs channel insensitive to these activators. Our experiments showed that the effect of R-L3 on voltage sensor movement during channel deactivation was much more prominent than that of ML277. Simulations using a KCNQ1 kinetic model showed that the effects of ML277 and R-L3 could be reproduced through two different effects on channel gating: ML277 enhances KCNQ1 channel function through a pore-dependent and voltage sensor-independent mechanism, while R-L3 affects both channel pore and voltage sensor.

Exploring mutation specific beta blocker pharmacology of the pathogenic late sodium channel current from patient-specific pluripotent stem cell myocytes derived from long QT syndrome mutation carriers.

The congenital long QT syndrome (LQTS), one of the most common cardiac channelopathies, is characterized by delayed ventricular repolarization underlying prolongation of the QT interval of the surface electrocardiogram. LQTS is caused by mutations in genes coding for cardiac ion channels or ion channel-associated proteins. The major therapeutic approach to LQTS management is beta blocker therapy which has been shown to be effective in treatment of LQTS variants caused by mutations in K+ channels. However, this approach has been questioned in the treatment of patients identified as LQTS variant 3(LQT3) patients who carry mutations in SCN5A, the gene coding for the principal cardiac Na+ channel. LQT3 mutations are gain of function mutations that disrupt spontaneous Na+ channel inactivation and promote persistent or late Na+ channel current (INaL) that delays repolarization and underlies QT prolongation. Clinical investigation of patients with the two most common LQT3 mutations, the ΔKPQ and the E1784K mutations, found beta blocker treatment a useful therapeutic approach for managing arrhythmias in this patient population. However, there is little experimental data that reveals the mechanisms underlying these antiarrhythmic actions. Here, we have investigated the effects of the beta blocker propranolol on INaL expressed by ΔKPQ and E1784K channels in induced pluripotent stem cells derived from patients carrying these mutations. Our results indicate that propranolol preferentially inhibits INaL expressed by these channels suggesting that the protective effects of propranolol in treating LQT3 patients is due in part to modulation of INaL.

The ß1'-ß2' Motif of the RNase H Domain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Is Responsible for Conferring Open Conformation to the p66 Subunit by Displacing the Connection Domain from the Polymerase Cleft.

The heterodimeric human immunodeficiency virus type 1 reverse transcriptase is composed of p66 and p51 subunits. While in the p51 subunit, the connection domain is tucked in the polymerase cleft; it is effectively displaced from the cleft of the catalytically active p66 subunit. How is the connection domain relocated from the polymerase cleft of p66? Does the RNase H domain have any role in this process? To answer this question, we extended the C-terminal region of p51 by stepwise addition of N-terminal motifs of RNase H domain to generate p54, p57, p60, and p63 derivatives. We found all of the C-terminal extended derivatives of p51 assume open conformation, bind to the template-primer, and catalyze the polymerase reaction. Glycerol gradient ultracentrifugation analysis showed that only p54 sedimented as a monomer, while other derivatives were in a homodimeric conformation. We proposed a model to explain the monomeric conformation of catalytically active p54 derivative carrying additional 21-residues long ß1'-ß2' motif from the RNase H domain. Our results indicate that the ß1'-ß2' motif of the RNase H domain may be responsible for displacing the connection domain from the polymerase cleft of putative monomeric p66. The unstable elongated p66 molecule may then readily dimerize with p51 to assume a stable dimeric conformation.

The glutamine side chain at position 91 on the ß5a-ß5b loop of human immunodeficiency virus type 1 reverse transcriptase is required for stabilizing the dNTP binding pocket.

Earlier, we postulated that Gln91 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) stabilizes the side chain of Tyr183 via hydrogen bonding interaction between O(H) of Tyr183 and CO of Q91 [Harris, D., et al. (1998) Biochemistry 37, 9630-9640]. To test this hypothesis, we generated mutant derivatives of Gln91 and analyzed their biochemical properties. The efficiency of reverse transcription was severely impaired by nonconservative substitution of Gln with Ala, while conservative substitution of Gln with Asn resulted in an approximately 70% loss of activity, a value similar to that observed with the Y183F mutation. The loss of polymerase activity from both Q91A and Q91N was significantly improved by a Met to Val substitution at position 184. Curiously, the Q91N mutant exhibited stringency in discriminating between correct and incorrect nucleotides, suggesting its possible interaction with residues influencing the flexibility of the dNTP binding pocket. In contrast, both double mutants, Q91A/M184V and Q91N/M184V, are found to be as error prone as the wild-type enzyme. We propose a model that suggests that subtle structural changes in the region due to mutation at position 91 may influence the stability of the side chain of Tyr183 in the catalytic YMDD motif of the enzyme, thus altering the active site geometry that may interfere in substrate recognition.