Showing papers by "Kenneth A. Stauderman published in 1999"

Functional Consequences of Mutations in the Human α1A Calcium Channel Subunit Linked to Familial Hemiplegic Migraine

Abstract: Mutations in α1A, 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 α1A-2subunits 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.

Structure and functional characterization of a novel human low-voltage activated calcium channel.

Abstract: 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.

Fluorescence techniques for measuring ion channel activity.

Abstract: Publisher Summary Changes in intracellular free calcium concentration ([Ca 2+ ] i ) play a crucial role in cellular physiology. A number of cell surface receptors and channels are known to regulate [Ca 2+ ] i through different molecular mechanisms. Therefore, the functional and pharmacologic properties of many of these cell surface receptors and ion channels can be studied effectively by measuring changes in [Ca 2+ ] i in intact cells. For drug discovery efforts, several ion channel and receptor systems have been targeted that play different roles in neuronal physiology and pathophysiology. These molecular targets include voltage- and ligand-gated ion channels: the human neuronal voltage-gated calcium channels (VGCCs), ligand-gated nicotinic acetylcholine receptor channels (NAChRs), ionotropic N -methyl-D-aspartic acid (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)- and kainate-type excitatory amino acid receptor (EAA) channels. All of these channels mediate elevation of [Ca 2+ ] i via Ca 2+ influx from the extracellular medium upon depolarization or activation by agonist. This chapter describes the experimental methods with particular emphasis on the validation of the assay for human VGCCs, NAChRs, and NMDA receptors.