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Biotech / Medical : SIBIA Neurosciences (SIBI) -- Ignore unavailable to you. Want to Upgrade?

To: scaram(o)uche who wrote (298)	3/27/1999 11:38:00 AM
From: scaram(o)uche	Respond to of 579

Biophys J 1999 Mar;76(3):1384-1400 Structural Elements in Domain IV that Influence Biophysical and Pharmacological Properties of Human alpha1A-Containing High-Voltage-Activated Calcium Channels. Hans M, Urrutia A, Deal C, Brust PF, Stauderman K, Ellis SB, Harpold MM, Johnson EC, Williams ME SIBIA Neurosciences, Inc., La Jolla, California 92037-4641 USA. [Record supplied by publisher] We have cloned two splice variants of the human homolog of the alpha1A subunit of voltage-gated Ca2+ channels. The sequences of human alpha1A-1 and alpha1A-2 code for proteins of 2510 and 2662 amino acids, respectively. Human alpha1A-2alpha2bdeltabeta1b Ca2+ channels expressed in HEK293 cells activate rapidly (tau+10mV = 2.2 ms), deactivate rapidly (tau-90mV = 148 mus), inactivate slowly (tau+10mV = 690 ms), and have peak currents at a potential of +10 mV with 15 mM Ba2+ as charge carrier. In HEK293 cells transient expression of Ca2+ channels containing alpha1A/B(f), an alpha1A subunit containing a 112 amino acid segment of alpha1B-1 sequence in the IVS3-IVSS1 region, resulted in Ba2+ currents that were 30-fold larger compared to wild-type (wt) alpha1A-2-containing Ca2+ channels, and had inactivation kinetics similar to those of alpha1B-1-containing Ca2+ channels. Cells transiently transfected with alpha1A/B(f)alpha2bdeltabeta1b expressed higher levels of the alpha1, alpha2bdelta, and beta1b subunit polypeptides as detected by immunoblot analysis. By mutation analysis we identified two locations in domain IV within the extracellular loops S3-S4 (N1655P1656) and S5-SS1 (E1740) that influence the biophysical properties of alpha1A. alpha1AE1740R resulted in a threefold increase in current magnitude, a -10 mV shift in steady-state inactivation, and an altered Ba2+ current inactivation, but did not affect ion selectivity. The deletion mutant alpha1ADeltaNP shifted steady-state inactivation by -20 mV and increased the fast component of current inactivation twofold. The potency and rate of block by omega-Aga IVA was increased with alpha1ADeltaNP. These results demonstrate that the IVS3-S4 and IVS5-SS1 linkers play an essential role in determining multiple biophysical and pharmacological properties of alpha1A-containing Ca2+ channels.

To: scaram(o)uche who wrote (298)	3/27/1999 11:39:00 AM
From: scaram(o)uche	Respond to of 579

J Neurosci 1999 Mar 1;19(5):1610-9 Functional consequences of mutations in the human alpha1A calcium channel subunit linked to familial hemiplegic migraine. Hans M, Luvisetto S, Williams ME, Spagnolo M, Urrutia A, Tottene A, Brust PF, Johnson EC, Harpold MM, Stauderman KA, Pietrobon D SIBIA Neurosciences, La Jolla, California 92037-4641, USA. [Medline record in process] Mutations in alpha1A, the pore-forming subunit of P/Q-type calcium channels, are linked to several human diseases, including familial hemiplegic migraine (FHM). We introduced the four missense mutations linked to FHM into human alpha1A-2 subunits and investigated their functional consequences after expression in human embryonic kidney 293 cells. By combining single-channel and whole-cell patch-clamp recordings, we show that all four mutations affect both the biophysical properties and the density of functional channels. Mutation R192Q in the S4 segment of domain I increased the density of functional P/Q-type channels and their open probability. Mutation T666M in the pore loop of domain II decreased both the density of functional channels and their unitary conductance (from 20 to 11 pS). Mutations V714A and I1815L in the S6 segments of domains II and IV shifted the voltage range of activation toward more negative voltages, increased both the open probability and the rate of recovery from inactivation, and decreased the density of functional channels. Mutation V714A decreased the single-channel conductance to 16 pS. Strikingly, the reduction in single-channel conductance induced by mutations T666M and V714A was not observed in some patches or periods of activity, suggesting that the abnormal channel may switch on and off, perhaps depending on some unknown factor. Our data show that the FHM mutations can lead to both gain- and loss-of-function of human P/Q-type calcium channels.

To: scaram(o)uche who wrote (298)	3/27/1999 11:47:00 AM
From: scaram(o)uche	Respond to of 579

J Neurochem 1999 Feb;72(2):791-9 Structure and functional characterization of a novel human low-voltage activated calcium channel. Williams ME, Washburn MS, Hans M, Urrutia A, Brust PF, Prodanovich P, Harpold MM, Stauderman KA SIBIA Neurosciences Inc., La Jolla, California 92037, USA. We have isolated and characterized overlapping cDNAs encoding a novel, voltage-gated Ca2+ channel alpha1 subunit, alpha1H, from a human medullary thyroid carcinoma cell line. The alpha1H subunit is structurally similar to previously described alpha1 subunits. Northern blot analysis indicates that alpha1H mRNA is expressed throughout the brain, primarily in the amygdala, caudate nucleus, and putamen, as well as in several nonneuronal tissues, with relatively high levels in the liver, kidney, and heart. Ba2+ currents recorded from human embryonic kidney 293 cells transiently expressing alpha1H activated at relatively hyperpolarized potentials (-50 mV), rapidly inactivated (tau = 17 ms), and slowly deactivated. Similar results were observed in Xenopus oocytes expressing alpha1H. Single-channel measurements in human embryonic kidney 293 cells revealed a single-channel conductance of approximately 9 pS. These channels are blocked by Ni2+ (IC50 = 6.6 microM) and the T-type channel antagonists mibefradil (approximately 50% block at 1 microM) and amiloride (IC50 = 167 microM). Thus, alpha1H-containing channels exhibit biophysical and pharmacological properties characteristic of low voltage-activated, or T-type, Ca2+ channels.

To: scaram(o)uche who wrote (298)	3/27/1999 12:05:00 PM
From: scaram(o)uche	Read Replies (1) \| Respond to of 579

Title: PYRIDINE DERIVATIVES Publication date: 1999-01-21 Inventor(s): ALLGEIER HANS (DE); AUBERSON YVES (CH); BIOLLAZ MICHEL (CH); COSFORD NICHOLAS DAVID (US); GASPARINI FABRIZIO (CH); HECKENDORN ROLAND (CH); JOHNSON EDWIN CARL (US); KUHN RAINER (DE); VARNEY MARK ANDREW (US); VELICELEBI GOENUEL (US) Applicant(s): NOVARTIS AG (CH); NOVARTIS ERFINDUNGEN VERWALTUN (AT); SIBIA NEUROSCIENCES INC (US); ALLGEIER HANS (DE); AUBERSON YVES (CH); BIOLLAZ MICHEL (CH); COSFORD NICHOLAS DAVID (US); GASPARINI FABRIZIO (CH); HECKENDORN ROLAND (CH); JOHNSON EDWIN CARL (US); KUHN RAINER (DE); VARNEY MARK ANDREW (US) Requested Patent: WO9902497 A 19990121 Application Number: WO1998EP04266 19980709 Priority Number(s): US19970891691 19970711; US19970890689 19970711 IPC Classification: C07D213/00