No abstract

Voltage-dependent inhibition by 1,4-dihydropyridines is a characteristic property of L-type Ca2+ channels. Six out of 50 exons of the channel alpha 1C subunit gene are subjected to alternative splicing, thus generating channel isoform diversity. Using Xenopus oocytes as an expression system, we have found that transmembrane segment IIIS2 of human alpha 1C subunit is involved in the control of voltage dependence of dihydropyridine action. This segment is genetically regulated through alternative splicing of exons 21/22. Site-directed mutagenesis points to two amino acids in IIIS2, which determine the difference of the splice variants in their sensitivities to dihydropyridines. This finding provides new insight into molecular mechanisms of Ca2+ channel inhibition by this important class of drugs.

(2). The channels are also designated as L-type and are multisubunit proteins composed of the poreforming ␣ 1C subunit, which contains high affinity binding sites for DHPs (3-7), and of the auxiliary ␤ and ␣ 2 ␦ subunits (8, 9). Analysis of the hydrophobicity profile of ␣ 1C indicates four repetitive motifs of similarity (I-IV), each composed of six transmembrane segments (S1-S6) (10). Both, the short aminoterminal tail encoded by exons 1 and 2, and the long carboxylterminal tail encoded by exons 38 -50 of the human ␣ 1C gene (11) are located in the cytoplasm.Expression of ␣ 1C is regulated through alternative splicing (12), which has primarily been detected in the membranespanning regions of the molecule. However, there is evidence that the carboxyl-terminal tail is also affected by alternative splicing. Two partial transcripts have been identified in a cDNA library of human hippocampus (11, 13). They show that exons 40 -43 encoding the second quarter of the putative cytoplasmic tail of the ␣ 1C molecule are subject to alternative splicing and may give rise to new ␣ 1C splice variants in the brain.The functional role of the carboxyl-terminal tail attracts much attention because of its potential involvement in channel gating. Removal of approximately 70% of the tail causes an increase in the opening probability of the rabbit cardiac ␣ 1C channel (14). A similar deletion mutant of the human cardiac ␣ 1C showed faster inactivation of the channel as compared to the wild-type channel (15). It has been concluded that this tail part of ␣ 1C may serve as a critical component of the gating structure that influences inactivation properties of the channel (15).In this report we describe two recombinant plasmids, pHLCC72 and pHLCC86, which contain alternative exons encoding parts of the carboxyl-terminal tails that are found in human hippocampus transcripts. After expression in Xenopus oocytes, we have analyzed electrophysiological properties of ␣ 1C,72 and ␣ 1C,86 channels and compared them with the reference ␣ 1C,77 channel (16). The results of our study show that amino acids encoded by exons 40 -42 are important for the voltage dependence of activation and inactivation of the current through these channels, as well as for the kinetics and Ca 2ϩ dependence of inactivation. MATERIALS AND METHODSPreparation of cDNAs Encoding ␣ 1C Subunit Splice Variants-All splice variants were constructed within the frame of pHLCC77 (16) composed of exons 1-20, 22-30, 32-44, and 46 -50 using the pBluescript SK(Ϫ) vector (Stratagene) flanked at the 5Ј-end with HindIII/ BglII and at the 3Ј-end with BglII/BamHI fragments of the Xenopus ␤-globin gene untranslated region sequences, respectively (17, 18). The recombinant plasmid pHLCC86 was prepared by replacing nucleotides 5104 -5482 of pHLCC77, encoding exons 41 and 42, with the BsaI/BglII

Kainate receptors (KARs) are abundantly expressed in the basal ganglia, but their function in synaptic transmission has not been established. In the present study, we show that the GluR6 subunit of KARs is expressed in both substance P- and enkephalin-containing GABAergic projection neurons of the mouse striatum. Using whole-cell voltage-clamp recordings in brain slices, we demonstrate the presence of functional KARs in the dorsal striatum activated by low concentrations of the AMPA/KAR agonist domoate in wild-type but not GluR6-deficient mice. Despite the abundance of KARs, we found no evidence for synaptic activation of these receptors after single or repetitive stimulation of glutamatergic afferents. Domoate induces a transient increase in the frequency of spontaneous IPSCs of small amplitude and a sustained depression of large IPSCs evoked by minimal electrical stimulation within the striatum in wild-type mice but not in GluR6-deficient mice. This depressant effect is inhibited in presence of adenosine A(2A) receptor antagonists, ZM-241385 and SCH-58261. These data strongly suggest that, in striatal neurons, KARs depress GABAergic synaptic transmission indirectly via release of adenosine acting on A(2A) receptors.

Transient receptor potential (TRP) channels form a diverse family of cation channels comprising 28 members in mammals. Although some TRP proteins can only be found on intracellular membranes, most of the TRP protein isoforms reach the plasma membrane where they form ion channels and control a wide number of biological processes. There, their involvement in the transport of cations such as calcium and sodium has been well documented. However, a growing number of studies have started to expand our understanding of these proteins by showing that they also transport other biologically relevant metal ions like zinc, magnesium, manganese and cobalt. In addition to this newly recognized property, the activity and expression of TRP channels can be regulated by metal ions like magnesium, gadolinium, lanthanum or cisplatin. The aim of this review is to highlight the complex relationship between metal ions and TRP channels.