Functional Consequences of Mutations in the Human α_1ACalcium Channel Subunit Linked to Familial Hemiplegic Migraine

Abstract

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.

Familial Hemiplegic Migraine

“…However, an important consistent effect was revealed by studying the single channel properties of eight mutant human Ca V 2.1 channels. All the FHM1 mutants showed an enhanced single channel Ca 2ϩ influx in a wide range of mild depolarizations, reflecting an increased channel open probability, mainly due to a shift to lower voltages of channel activation 31,34,35 (and our unpublished observations). Consistent (and in the majority of cases significant) shifts to lower voltages of current activation of human FHM1 mutants were also revealed by measurements of whole-cell currents in heterologous expression systems, transfected neurons and neurons of FHM1 knockin mice.…”

“…By contrast, varying and conflicting results were obtained from previous measurements of the whole-cell Ca 2ϩ current in transfected neurons and HEK293 cells overexpressing mutant human Ca V 2.1 channels. While either an increase or a decrease of the whole cell Ca V 2.1 current density elicited by strong depolarizations was measured in HEK293 cells depending on the FHM1 mutant analyzed 31,34,35 (and our unpublished observations), a consistent decrease was measured in neurons. 31,35,37,38 The R192Q mutation decreased the current density at high voltages in transfected neurons but increased it in transfected HEK293 cells.…”

“…Consistent (and in the majority of cases significant) shifts to lower voltages of current activation of human FHM1 mutants were also revealed by measurements of whole-cell currents in heterologous expression systems, transfected neurons and neurons of FHM1 knockin mice. 30,31,[34][35][36][39][40][41] The Ca V 2.1 current density in cerebellar granule cells and cortical pyramidal neurons of R192Q and S218L knockin mice was larger than in wild-type neurons for a wide range of mild depolarizations. Similar current densities were measured with strong depolarizations (eliciting maximal channel open probability) indicating a similar number of functional Ca V 2.1 channels in the membrane of knockin and wild-type neurons.…”

“…31,35,37,38 The R192Q mutation decreased the current density at high voltages in transfected neurons but increased it in transfected HEK293 cells. 31,34,37,38 Because the same mutation did not affect the whole-cell P/Q Ca 2ϩ current density elicited by strong depolarizations in neurons of knockin mice expressing Ca V 2.1 channels at their endogenous level, the changes in transfected cells likely reflect changes in the number of functional channels in the membrane that might be an artifact of overexpression. One may therefore obtain misleading answers using transfected cells to study the effect of ion channel mutations on cellular functions that depend on the number of functional channels in the membrane.…”

Physiologic Principles Underlying Ion Channelopathies

“…Some missense mutations in the Cav2.1 P/Q-type Ca 2ϩ channel that occur in familial hemiplegic migraine have a reduced unitary conductance. 23 Autosomal dominant nocturnal frontal lobe epilepsy results from a serine to phenylalanine mutation in the pore region (M2) of the ␣ 4 nicotinic acetylcholine receptor subunit that alters both permeation and gating. In mutant channels, the single-channel conductance is reduced nearly twofold, permeability to Ca 2ϩ is lost, desensitization occurs more rapidly, and recovery from desensitization is prolonged.…”