Publications (2)13.79 Total impact
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Article: Structure and inhibition of the SARS coronavirus envelope protein ion channel.
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ABSTRACT: The envelope (E) protein from coronaviruses is a small polypeptide that contains at least one alpha-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA), but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV) that the transmembrane domain of E protein (ETM) forms pentameric alpha-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular alpha-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293) cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA), but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target.PLoS Pathogens 08/2009; 5(7):e1000511. · 9.13 Impact Factor -
Article: Differential splicing patterns of L-type calcium channel Cav1.2 subunit in hearts of Spontaneously Hypertensive Rats and Wistar Kyoto Rats.
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ABSTRACT: Cav1.2 L-type calcium channels are essential in heart and smooth muscle contraction. Rat Cav1.2 gene contains 11 alternatively spliced exons (1a, 1, 8a, 8, 9*, 21, 22, 31, 32, 32-6nt and 33) which can be assorted to generate a large number of functionally distinct splice variants. Until now, it is unknown whether the utilization of these alternatively spliced exons is altered in the hypertrophied hearts of hypertensive rats. By comparing the assortments of these 11 exons in full-length Cav1.2 transcripts derived from Spontaneously Hypertensive Rats (SHRs) and Wistar Kyoto Rats (WKYs) hearts, we found that the inclusion of Cav1.2 alternative exons was significantly different between the two rats both at individual loci and in combinatorial arrangements. Functional characterizations of three Cav1.2 channel splice variants that were identified to be significantly altered in SHR hypertrophied cardiomyocytes demonstrated distinct whole-cell electrophysiological properties when expressed in HEK 293 cells. Interestingly, aberrant splice variants which included or excluded both mutually exclusive exons 21/22 or exons 31/32 were found to be increased in hypertensive rats. Two aberrant splice variants that included both exons 21 and 22 were found to be unable to conduct currents even though they expressed proteins with the predicted molecular mass. Characterization of one of the aberrant splice variants showed that it exerted a dominant negative effect on the functional Cav1.2 channels when co-expressed in HEK293 cells. The altered combinatorial splicing profiles of Cav1.2 transcripts identified in SHR hearts provide a different and new perspective in understanding the possible role of molecular remodeling of Cav1.2 channels in cardiac hypertrophy as a consequence of hypertension.Biochimica et Biophysica Acta 02/2008; 1783(1):118-30. · 4.66 Impact Factor
