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.

Expression of functional, recombinant ␣7 nicotinic acetylcholine receptors in several mammalian cell types, including HEK293 cells, has been problematic. We have isolated the recently described human ric-3 cDNA and co-expressed it in Xenopus oocytes and HEK293 cells with the human nicotinic acetylcholine receptor ␣7 subunit. In addition to confirming the previously reported effect on ␣7 receptor expression in Xenopus oocytes we demonstrate that ric-3 promotes the formation of functional ␣7 receptors in mammalian cells, as determined by whole cell patch clamp recording and surface ␣-bungarotoxin binding. Upon application of 1 mM nicotine, currents were undetectable in HEK293 cells expressing only the ␣7 subunit. In contrast, co-expression of ␣7 and ric-3 cDNAs resulted in currents that averaged 42 pA/pF with kinetics similar to those observed in cells expressing endogenous ␣7 receptors. Immunoprecipitation studies demonstrate that ␣7 and ric-3 proteins co-associate. Additionally, cell surface labeling with biotin revealed the presence of ␣7 protein on the plasma membrane of cells lacking ric-3, but surface ␣-bungarotoxin staining was only observed in cells co-expressing ric-3. Thus, ric-3 appears to be necessary for proper folding and/or assembly of ␣7 receptors in HEK293 cells. Nicotinic acetylcholine receptors (nAChRs)1 are members of the neurotransmitter-gated ion channel superfamily. They are widely expressed in the central and peripheral nervous system (1) where they influence numerous cellular and physiological processes. At least 17 different genes that code for nAChR subunits have been identified (2, 3), and they assemble as pentamers in different combinations to form a diverse set of nAChR subtypes (4, 5). The simplest case is the homopentameric complex such as that formed by the nAChR ␣7 subunit. The ␣7 receptor, for which ␣-bungarotoxin (␣-Bgt) is a specific and high affinity antagonist, is one of the most abundant receptor subtypes in the mammalian brain (6, 7). The high Ca 2ϩ permeability of the ␣7 receptor (8) suggests an involvement in the activation of Ca 2ϩ -dependent events in neurons such as transmitter release, participation in signal transduction, and a variety of modulatory effects (9). In addition, ␣7 receptors have been implicated in a number of diseases such as schizophrenia, Alzheimers, and Parkinsons disease (1, 10 -12).Heterologous expression of the ␣7 subunit in Xenopus oocytes results in homooligomeric, ␣-Bgt-sensitive receptors that activate and inactivate quickly and are highly permeable to Ca 2ϩ (8,13,14), similar to the properties of ␣7 nAChRs in neuronal cells. Although there have been reports of successful functional expression in some mammalian cell lines (15-18), measurable levels of functional receptors have been difficult to achieve in multiple cell types and this phenomenon appears to be host-cell dependent (19). The reasons for poor heterologous surface expression in these cells are not well understood. Strategies to increase the number of functional receptors on the cell...

Abstract:We have isolated and characterized overlapping cDNAs encoding a novel, voltage-gated Ca 2ϩ channel ␣ 1 subunit, ␣ 1H , from a human medullary thyroid carcinoma cell line. The ␣ 1H subunit is structurally similar to previously described ␣ 1 subunits. Northern blot analysis indicates that ␣ 1H 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. Ba 2ϩ currents recorded from human embryonic kidney 293 cells transiently expressing ␣ 1H activated at relatively hyperpolarized potentials (Ϫ50 mV), rapidly inactivated ( ϭ 17 ms), and slowly deactivated. Similar results were observed in Xenopus oocytes expressing ␣ 1H . Singlechannel measurements in human embryonic kidney 293 cells revealed a single-channel conductance of ϳ9 pS. These channels are blocked by Ni 2ϩ (IC 50 ϭ 6.6 M) and the T-type channel antagonists mibefradil (ϳ50% block at 1 M) and amiloride (IC 50 ϭ 167 M). Thus, ␣ 1H -containing channels exhibit biophysical and pharmacological properties characteristic of low voltage-activated, or T-type, Ca 2ϩ channels.

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 micros), 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.

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