Sequencing of the T-type Ca2ϩ channel gene CACNA1H revealed 12 nonsynonymous single nucleotide polymorphisms (SNPs) that were found only in childhood absence epilepsy (CAE) patients. One SNP, G773D, was found in two patients. The present study reports the finding of a third patient with this SNP, as well as analysis of their parents. Because of the role of T-channels in determining the intrinsic firing patterns of neurons involved in absence seizures, it was suggested that these SNPs might alter channel function. The goal of the present study was to test this hypothesis by introducing these polymorphisms into a human Ca v 3.2a cDNA and then study alterations in channel behavior using whole-cell patch-clamp recording. Eleven SNPs altered some aspect of channel gating. Computer simulations predict that seven of the SNPs would increase firing of neurons, with three of them inducing oscillations at similar frequencies, as observed during absence seizures. Three SNPs were predicted to decrease firing. Some CAE-specific SNPs (e.g., G773D) coexist with SNPs also found in controls (R788C); therefore, the effect of these polymorphisms were studied. The R788C SNP altered activity in a manner that would also lead to enhanced burst firing of neurons. The G773D-R788C combination displayed different behavior than either single SNP. Therefore, common polymorphisms can alter the effect of CAE-specific SNPs, highlighting the importance of sequence background. These results suggest that CACNA1H is a susceptibility gene that contributes to the development of polygenic disorders characterized by thalamocortical dysrhythmia, such as CAE.

Calcium currents via low-voltage-activated T-type channels mediate burst firing, particularly in thalamic neurons. Considerable evidence supports the hypothesis that overactive T-channels may contribute to thalamocortical dysrhythmia, including absence epilepsy. Single nucleotide polymorphisms in one of the T-channel genes (CACNA1H, which encodes Ca v 3.2) are associated with childhood absence epilepsy in a Chinese population. Because only a fraction of these polymorphisms are predicted to increase channel activity and neuronal firing, we hypothesized that other channel properties may be affected. Here we describe that all the polymorphisms clustered in the intracellular loop connecting repeats I and II (I-II loop) increase the surface expression of extracellularly tagged Ca v 3.2 channels. The functional domains within the I-II loop were then mapped by deletion analysis. The first 62 amino acids of the loop (post IS6) are involved in regulating the voltage dependence of channel gating and inactivation. Similarly, the last 15 amino acids of the loop (pre IIS1) are involved in channel inactivation. In contrast, the central region of I-II loop regulates surface expression, with no significant effect on channel biophysics. Electrophysiology, luminometry, fluorescence-activated cell sorting measurements, and confocal microscopy studies demonstrate that deletion of this central region leads to enhanced surface expression of channels from intracellular compartments to the plasma membrane. These results provide novel insights into how CACNA1H polymorphisms may contribute to Ca V 3.2 channel overactivity and consequently to absence epilepsy and establish the I-II loop as an important regulator of Ca V 3.2 channel function and expression.

High voltage-activated Ca 2+ channel expression and gating is controlled by their b subunits. Although the sites of interaction are known at the atomic level, how b modulates gating remains to be determined. Using a chimeric approach, b subunit regulation was conferred to a low voltage-activated channel. Regulation was dependent on a rigid linker connecting the a 1 interaction domain to IS6. Chimeric channels also revealed a role for IS6 in channel gating. Taken together, these results support a direct coupling model where b subunits alter movements in IS6 that occur as the channel transits between closed, open, and inactivated states.

BackgroundThe Cavβ subunits of high voltage-activated Ca2+ channels control the trafficking and biophysical properties of the α1 subunit. The Cavβ-α1 interaction site has been mapped by crystallographic studies. Nevertheless, how this interaction leads to channel regulation has not been determined. One hypothesis is that βs regulate channel gating by modulating movements of IS6. A key requirement for this direct-coupling model is that the linker connecting IS6 to the α-interaction domain (AID) be a rigid structure.Methodology/Principal FindingsThe present study tests this hypothesis by altering the flexibility and orientation of this region in α12.2, then testing for Cavβ regulation using whole cell patch clamp electrophysiology. Flexibility was induced by replacement of the middle six amino acids of the IS6-AID linker with glycine (PG6). This mutation abolished β2a and β3 subunits ability to shift the voltage dependence of activation and inactivation, and the ability of β2a to produce non-inactivating currents. Orientation of Cavβ with respect to α12.2 was altered by deletion of 1, 2, or 3 amino acids from the IS6-AID linker (Bdel1, Bdel2, Bdel3, respectively). Again, the ability of Cavβ subunits to regulate these biophysical properties were totally abolished in the Bdel1 and Bdel3 mutants. Functional regulation by Cavβ subunits was rescued in the Bdel2 mutant, indicating that this part of the linker forms β-sheet. The orientation of β with respect to α was confirmed by the bimolecular fluorescence complementation assay.Conclusions/SignificanceThese results show that the orientation of the Cavβ subunit relative to the α12.2 subunit is critical, and suggests additional points of contact between these subunits are required for Cavβ to regulate channel activity.

We have combined patch clamp recording with simultaneous [Ca2+]i measurements in single LNCaP cells (a human prostate cancer cell line), to study the activation of Ca2+‐permeable channels by two different inducers of apoptosis, ionomycin and serum deprivation. In perforated patch recording, LNCaP cells had a membrane potential of ‐40 mV and a resting [Ca2+]i of 90 nM. Application of ionomycin at levels that induced apoptosis in these cells (10 μM) produced a biphasic increase in [Ca2+]i. The first rise in [Ca2+]i was due to release of Ca2+ from internal stores and it was associated with a membrane hyperpolarization to ‐77 mV. The latter was probably due to the activation of high conductance, Ca2+‐ and voltage‐dependent K+ channels (maxi‐K). Conversely, the second rise in [Ca2+]i was always preceded by and strictly associated with membrane depolarization and required external Ca2+. Serum deprivation, another inducer of apoptosis, unmasked a voltage‐independent Ca2+ permeability as well. A lower concentration of ionomycin (1 μM) did not induce apoptosis, and neither depolarized LNCaP cells nor produced the biphasic increase in [Ca2+]i. However, the first increment in [Ca2+]i due to release from internal Ca2+ stores was evident at this concentration of ionomycin. Simultaneous recordings of [Ca2+]i and ion channel activity in the cell attached configuration of patch clamp revealed a Ca2+‐permeable, Ca2+‐independent, non‐selective cation channel of 23 pS conductance. This channel was activated only during the second increment in [Ca2+]i induced by ionomycin. The absence of serum activated the 23 pS channel as well, albeit at a lower frequency than with ionomycin. Thus, the 23 pS channel can be activated by two unrelated inducers of apoptosis and it could be another Ca2+ influx mechanism in programmed cell death of LNCaP cells.