A mutation in the first intracellular loop of CACNA1A prevents P/Q channel modulation by SNARE proteins and lowers exocytosis

Abstract

Familial hemiplegic migraine (FHM)-causing mutations in the gene encoding the P/Q Ca 2+ channel α 1A subunit (CACNA1A) locate to the pore and voltage sensor regions and normally involve gainof-channel function. We now report on a mutation identified in the first intracellular loop of CACNA1A (α 1A(A454T) ) that does not cause FHM but is associated with the absence of sensorimotor symptoms in a migraine with aura pedigree. α 1A(A454T) channels showed weakened regulation of voltage-dependent steady-state inactivation by Ca V β subunits. More interestingy, A454T mutation suppressed P/Q channel modulation by syntaxin 1A or SNAP-25 and decreased exocytosis. Our findings reveal the importance of I-II loop structural integrity in the functional interaction between P/Q channel and proteins of the vesicle-docking/fusion machinery, and that genetic variation in CACNA1A may be not only a cause but also a modifier of migraine phenotype.CaV 2.1 (P/Q) channels | SNARE proteins | migraine with aura F amilial hemiplegic migraine (FHM) is an autosomal dominantly inherited subtype of migraine with aura that features some degree of hemiparesis during attacks (1, 2). The generally accepted view on migraine pathophysiology points to cortical spreading depression (CSD), an abnormal increase of cortical activity-followed by a long-lasting neuronal suppression wavethat propagates across the cortex, as the cause of the aura and migraine itself (1, 3). FHM-causing mutations have been reported in the CACNA1A gene (encoding the P/Q Ca 2+ channel α 1 subunit) (4), resulting in a gain of P/Q channel function, mainly due to a reduction in the voltage threshold of channel activation favoring CSD initiation and propagation (1, 5-11). Other genetic and environmental factors may also play a role in shaping the phenotype, as identical mutations show different clinical characteristics (2).The P/Q Ca 2+ channel contains a pore-forming α 1A subunit and several regulatory subunits, including intracellular β subunits (Ca V β 1-4 ) that bind to the intracellular loop between transmembrane domains I and II of α 1A (see Fig. 1B for an illustration of the channel complex). The effect of the regulatory subunits is essential for increasing the expression levels and modulating the voltage-dependent activation and inactivation of P/Q channels (12-15).Presynaptic proteins of the vesicle-docking/fusion machinery, including plasma membrane SNARE proteins (syntaxin 1A and SNAP-25) and synaptotagmin, bind to a specific site (synprint) in the large intracellular loop connecting domains II and III of the P/Q channel α 1A subunit (Fig. 1B). This interaction allows secretory vesicles docking to the plasma membrane near the pathway for Ca 2+ entry, optimizing neurotransmitter release.Syntaxin 1A and SNAP-25 also exert an inhibitory effect on P/Q channel activity by left-shifting the voltage dependence of steadystate inactivation (12,16,17). The synprint site serves an important anchoring function that may facilitate SNARE's modulation of channel gating, but the involvem...

A Cav3.2/Syntaxin-1A Signaling Complex Controls T-type Channel Activity and Low-threshold Exocytosis

“…Besides modulating T-type Ca 2ϩ channel gating, the physical association between Ca v 3.2 channel and SNARE proteins may contribute significantly to optimize Ca v 3.2-mediated exocytosis. Consistent with previous studies showing that Ca v 2.1, Ca v 2.2, or Ca v 3.1 channels can trigger voltage-dependent exocytosis when heterologously expressed in MPC-9/3L cells (6,31,48), we show that expression of Ca v 3.2 channel in these cells induced robust voltage-dependent exocytosis that was drastically reduced when SNARE proteins were competitively uncoupled from the channel by co-expression of the carboxylterminal domain of the Ca v 3.2 subunit. This result is consistent with previous studies showing that disruption of N-and P/Qtype Ca 2ϩ channel-SNARE interaction by a peptide containing the synprint site prevents neurotransmitter release (12,13).…”

“…Total charge transferred during each pulse was evaluated by integrating the area below gating current trace at the beginning (Q on ) and after the end of each depolarizing pulse (Q off ). For capacitance measurements, MPC 9/3L-AH cells were transfected using Lipofectamine Plus (Invitrogen), as previously reported (6,31 and had a resistance of 2-4 M⍀. Capacitance measurements were performed using an EPC-10 patch-clamp amplifier using the "sine ϩ dc" software lock-in amplifier method implemented in PatchMaster software.…”

TRPM5-mediated calcium uptake regulates mucin secretion from human colon goblet cells

“…The whole-cell configuration of the patch-clamp technique was employed as previously describe to test for the functional expression of TRP channel activity (Fernandes et al, 2008) and voltage-gated calcium currents (Serra et al, 2010). Pipettes with a resistance of 2–3 MΩ were used.…”

Screening of CACNA1A and ATP1A2 genes in hemiplegic migraine: clinical, genetic, and functional studies

“…Clinical variation is also seen within the HM phenotype, a condition in which CACNA1A may sometimes be implicated as a modifier gene rather than a disease‐causing gene (Serra et al. ). Typical attacks in HM are often associated with other aura symptoms: the clinical spectrum includes permanent cerebellar signs and, less frequently, various types of epileptic seizures, mental retardation, and coma.…”