No abstract

Properties of Ba2+ currents through voltage-dependent Ca2+ channels (IBa) were investigated in 61 dentate granule cells acutely isolated from the resected hippocampus of nine patients with therapy-refractory temporal lobe epilepsy (TLE). Currents with a high threshold of activation (HVA) peaked at 0 mV, and showed some time-dependent inactivation and a voltage of half-maximal steady-state inactivation (V1/2inact) of -16.4 mV. Application of saturating doses of omega-conotoxin (omega-CgTx) GVIA or nifedipine distinguished characteristic N-type (38%) and L-type (62% of HVA currents) Ca2+ currents. Combined application of both agents blocked HVA currents by > 95%. In a 10-mo-old child but not in adult patients, an omega-agatoxin IVA (omega-AgaTxIVA)-sensitive but omega-CgTx MVIIC-insensitive, noninactivating component of HVA currents (approximately 24%) was present that most probably corresponds to a P-type current. A T-type Ca2+ current could be separated from HVA components on the basis of its steady-state voltage-dependent inactivation (V1/2inact = -71.0 mV). The T-type Ca2+ current isolated by subtraction peaked at more negative potentials (-10 mV), showed a significantly more rapid time-dependent inactivation, and could be selectively blocked by low concentrations of Ni2+. It was insensitive to nifedipine and omega-CgTx GVIA. We conclude that L-, N-, and T-type currents are present in adult human dentate granule cells and an additional P-type current is present in neurons from a 10-mo-old patient. These data may provide a basis for comparison with animal models of epilepsy and for the elucidation of mechanisms of action of drugs intended for use in human disease.

Intracellular Ca(2+) represents an important trigger for various second-messenger mediated effects. Therefore a stringent control of the intracellular Ca(2+) concentration is necessary to avoid excessive activation of Ca(2+)-dependent processes. Ca(2+)-dependent inactivation of voltage-dependent calcium currents (VCCs) represents an important negative feedback mechanism to limit the influx of Ca(2+) that has been shown to be altered in the kindling model of epilepsy. We therefore investigated the Ca(2+)-dependent inactivation of high-threshold VCCs in dentate granule cells (DGCs) isolated from the hippocampus of patients with drug-refractory temporal lobe epilepsy (TLE) using the patch-clamp method. Ca(2+) currents showed pronounced time-dependent inactivation when no extrinsic Ca(2+) buffer was present in the patch pipette. In addition, in double-pulse experiments, Ca(2+) entry during conditioning prepulses caused a reduction of VCC amplitudes elicited during a subsequent test pulse. Recovery from Ca(2+)-dependent inactivation was slow and only complete after 1 s. Ca(2+)-dependent inactivation could be blocked either by using Ba(2+) as a charge carrier or by including bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) or EGTA in the intracellular solution. The influence of the cytoskeleton on Ca(2+)-dependent inactivation was investigated with agents that stabilize and destabilize microfilaments or microtubules, respectively. From these experiments, we conclude that Ca(2+)-dependent inactivation in human DGCs involves Ca(2+)-dependent destabilization of both microfilaments and microtubules. In addition, the microtubule-dependent pathway is modulated by the intracellular concentration of GTP, with lower concentrations of guanosine triphosphate (GTP) causing increased Ca(2+)-dependent inactivation. Under low-GTP conditions, the amount of Ca(2+)-dependent inactivation was similar to that observed in the kindling model. In summary, Ca(2+)-dependent inactivation was present in patients with TLE and Ammon's horn sclerosis (AHS) and is mediated by the cytoskeleton similar to rat pyramidal neurons. The similarity to the kindling model of epilepsy may suggest the possibility of altered Ca(2+)-dependent inactivation in patients with AHS.

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