Familial hemiplegic migraine type 1 mutations W1684R and V1696I alter G protein-mediated regulation of CaV2.1 voltage-gated calcium channels

Abstract

Familial hemiplegic migraine type 1 (FHM-1) is a monogenic form of migraine with aura that is characterized by recurrent attacks of a typical migraine headache with transient hemiparesis during the aura phase. In a subset of patients, additional symptoms such as epilepsy and cerebellar ataxia are part of the clinical phenotype. FHM-1 is caused by missense mutations in the CACNA1A gene that encodes the pore-forming subunit of Ca(V)2.1 voltage-gated Ca(2+) channels. Although the functional effects of an increasing number of FHM-1 mutations have been characterized, knowledge on the influence of most of these mutations on G protein regulation of channel function is lacking. Here, we explored the effects of G protein-dependent modulation on mutations W1684R and V1696I which cause FHM-1 with and without cerebellar ataxia, respectively. Both mutations were introduced into the human Ca(V)2.1α(1) subunit and their functional consequences investigated after heterologous expression in human embryonic kidney 293 (HEK-293) cells using patch-clamp recordings. When co-expressed along with the human μ-opioid receptor, application of the agonist [d-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) inhibited currents through both wild-type (WT) and mutant Ca(V)2.1 channels, which is consistent with the known modulation of these channels by G protein-coupled receptors. Prepulse facilitation, which is a way to characterize the relief of direct voltage-dependent G protein regulation, was reduced by both FHM-1 mutations. Moreover, the kinetic analysis of the onset and decay of facilitation showed that the W1684R and V1696I mutations affect the apparent dissociation and reassociation rates of the Gβγ dimer from the channel complex, suggesting that the G protein-Ca(2+) channel affinity may be altered by the mutations. These biophysical studies may shed new light on the pathophysiology underlying FHM-1.

The E1015K Variant in the Synprint Region of the CaV2.1 Channel Alters Channel Function and Is Associated with Different Migraine Phenotypes

“…In this altered conformation, SNARE binding to the synprint site may no longer exert the same forces that are required for inactivation of the channel. Similar studies have also shown that migraine mutations in Ca V 2.1 affect the influence of regulatory proteins including SNAREs (21), G proteins (38,39), and calmodulin-mediated facilitation (27).…”

Cav2.1 Channels and Migraine

“…Studies of recombinant human Ca v 2.1 channels carrying five different FHM1 mutations have shown that the migraine mutations reduce G-protein-mediated inhibitory modulation of Ca v 2.1 channels (Melliti et al 2003;Weiss et al 2008;Serra et al 2009;Garza-Lopez et al 2012), an effect that may lead to further gainof-function of Ca influx through mutant channels during neuromodulation. The only study that investigated voltage-dependent G-protein modulation using physiological-like stimuli revealed a reduced Gbc-mediated inhibition of the Ca current evoked by AP-like stimuli and a reduced facilitation of the Ca current carried by mutant channels after a train of short depolarizing pulses in cells overexpressing Gbc subunits; the reduction of G-protein modulation was dependent on the duration of the AP, being larger with APs of longer duration (Serra et al 2009).…”

“…1.1) shifts to lower voltages of activation of recombinant mutant channels were also revealed by measurements of whole-cell current in heterologous expression systems (Kraus et al 1998(Kraus et al , 2000Hans et al 1999;Tottene et al 2002Tottene et al , 2005Melliti et al 2003;Mullner et al 2004;Weiss et al 2008;Adams et al 2009;Serra et al 2009;Garza-Lopez et al 2012) and transfected neurons (where five FHM1 mutations, underlined in Fig. 1.1, were analyzed) (Tottene et al 2002(Tottene et al , 2005.…”

Studies on the Pathophysiology and Genetic Basis of Migraine

“…Despite the large number of mutations characterized at each FHM locus, only a small fraction of mutations that produce very extreme phenotypes have been studied in genetically altered mice given the expense and time involved in generating modified animals [58]. …”