To investigate the molecular basis of the calcium channel block by diltiazem, we transferred amino acids of the highly sensitive and stereoselective L-type (␣ 1S or ␣ 1C ) to a weakly sensitive, nonstereoselective class A (␣ 1A ) calcium channel. Transfer of three amino acids of transmembrane segment IVS6 of L-type ␣ 1 into the ␣ 1A subunit (I1804Y, S1808A, and M1811I) was sufficient to support a use-dependent block by diltiazem and by the phenylalkylamine (؊)-gallopamil after expression in Xenopus oocytes. An additional mutation F1805M increased the sensitivity for (؊)-gallopamil but not for diltiazem. Our data suggest that the receptor domains for diltiazem and gallopamil have common but not identical molecular determinants in transmembrane segment IVS6. These mutations also identified single amino acid residues in segment IVS6 that are important for class A channel inactivation.L-type calcium (Ca 2ϩ ) channels (classes C (formed by ␣ 1C subunits), D (␣ 1D ), and S (␣ 1S )) possess high affinity stereoselective drug receptors for Ca 2ϩ antagonists such as 1,4-dihydropyridines (DHPs), 1 phenylalkylamines (PAAs), and benzothiazepines (BTZs) (reviewed in Refs. 1-5) located on their pore-forming ␣ 1 channel subunit (6). Classes A (␣ 1A ), B (␣ 1B ), and E (␣ 1E ) Ca 2ϩ channels are insensitive for DHPs (2, 5, 7-9) and only weakly sensitive for . Essential parts of the high affinity binding sites for DHPs and PAAs on L-type Ca 2ϩ channels have been identified by replacing sequence stretches in ␣ 1C or ␣ 1S subunits by corresponding non-L-type sequences (8, 13) or by mutating single amino acids in these subunits (10, 13). Alternatively, molecular determinants of the high affinity DHP and PAA receptor sites could be localized in pore-lining regions of repeats III and/or IV by transferring L-type ␣ 1 sequences into the ␣ 1A subunit (9, 12). Transfer of segment IVS6 from ␣ 1S to ␣ 1A enhanced PAA sensitivity of the resulting ␣ 1A /␣ 1S chimera to the level of L-type ␣ 1 subunits (12).The efficacy of the BTZ diltiazem as an antiarrhythmic and antihypertensive drug is due to its voltage-and use-dependent block of L-type Ca 2ϩ channels (14). Studies on cloned ␣ 1 subunits of different Ca 2ϩ channel classes (C, B, A, and E) have enabled a more precise characterization of their pharmacological features (15). To identify the molecular determinants of the high affinity BTZ interaction domain of L-type Ca 2ϩ channels, we introduced corresponding L-type sequence stretches into ␣ 1A . The diltiazem sensitivity of the resulting ␣ 1 chimeras was measured as use-dependent barium current (I Ba ) block after coexpression with ␤ 1a (16) and ␣ 2 /␦ (17) in Xenopus oocytes. EXPERIMENTAL PROCEDURESMolecular Biology-The construction of L-type chimera Lh (repeats I-IV from ␣ 1C-a (18)) with the N terminus replaced with ␣ 1S (19), as well as construction of chimeras AL12h and AL22, were described previously (9, 12). Chimera AL20 was generated by replacing the ClaI (nucleotide position, 4925)-XbaI (3Ј-polylinker) fragment of AL9 (9) by ...

Syntaxin, a membrane protein vital in triggering vesicle fusion, interacts with voltage-gated N- and P/Q-type Ca(2+) channels. This biochemical association is proposed to colocalize Ca(2+) channels and presynaptic release sites, thus supporting rapid and efficient initiation of neurotransmitter release. The syntaxin channel interaction may also support a novel signaling function, to modulate Ca(2+) channels according to the state of the associated release machinery (Bezprozvanny et al., 1995; Wiser et al., 1996; see also Mastrogiacomo et al., 1994). Here we report that syntaxin 1A (syn1A) coexpressed with N-type channels in Xenopus oocytes greatly promoted slow inactivation gating, but had little or no effect on the onset of and recovery from fast inactivation. Accordingly, the effectiveness of syntaxin depended strongly on voltage protocol. Slow inactivation was found for N-type channels even in the absence of syntaxin and could be distinguished from fast inactivation on the basis of its slow kinetics, distinct voltage dependence (voltage-independent at potentials higher than the level of half-inactivation), and temperature independence (Q(10), approximately 0.8). Trains of action potential-like stimuli were more effective than steady depolarizations in stabilizing the slowly inactivated condition. Agents that stimulate protein kinase C decreased the inhibitory effect of syntaxin on N-type channels. Application of BoNtC1 to cleave syntaxin sharply attenuated the modulatory effects on Ca(2+) channel gating, consistent with structural analysis of syntaxin modulation, supporting use of this toxin to test for the impact of syntaxin on Ca(2+) influx in nerve terminals.

The neuropeptide galanin is widely expressed in the central nervous system and other tissues and induces different cellular reactions, e.g. hormone release from pituitary and inhibition of insulin release from pancreatic B cells. By microinjection of antisense oligonucleotides we studied the question as to which G proteins mediate the galanin‐induced inhibition of voltage‐gated Ca2+ channels in the rat pancreatic B‐cell line RINm5F and in the rat pituitary cell line GH3. Injection of antisense oligonucleotides directed against alpha 01, beta 2, beta 3, gamma 2 and gamma 4 G protein subunits reduced the inhibition of Ca2+ channel current which was induced by galanin, whereas no change was seen after injection of cells with antisense oligonucleotides directed against alpha i, alpha q, alpha 11, alpha 14, alpha 15, beta 1, beta 4, gamma 1, gamma 3, gamma 5, or gamma 7 G protein subunits or with sense control oligonucleotides. In view of these data and of previous results, we conclude that the galanin receptors in GH3 and in RINm5F cells couple mainly to the G(0) protein consisting of alpha 01 beta 2 gamma 2 to inhibit Ca2+ channels and use alpha 01beta 3 gamma 4 less efficiently. The latter G protein composition was previously shown to be used by muscarinic M4 receptors to inhibit Ca2+ channels.

The effect of (R,S)-(3,4-dihydro 6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl- N,N-di-[2-(2,3,4-trimethoxyphenyl)ethyl]-acetamide (LOE 908), a cation channel blocker in HL-60 promyeloblasts, was studied in the A7r5 smooth muscle cell line from rat thoracic aorta, using the whole-cell patch-clamp technique. At a holding potential of -60 mV, application of vasopressin induced a nonselective cation conductance in voltage-clamped A7r5 cells. The current-voltage relation was linear, and currents reversed close to 0 mV regardless of the chloride gradient. The activation of the nonselective cation conductance by vasopressin was not affected by dialysing cells with Ca(2+)-free internal solution. LOE 908 blocked this current in a concentration-dependent manner with an IC50 of 560 nM, whereas dihydropyridine-sensitive Ba2+ current through voltage-dependent Ca2+ channels was blocked with an IC50 of 28 microM. Another organic blocker of receptor-mediated Ca2+ entry, 1-beta-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenethyl-1H-imidazole hydrochloride (SK&F 96365), blocked both, the vasopressin-induced nonselective conductance and the voltage-activated Ba2+ current with similar IC50 values of 13 microM and 8 microM, respectively. The rank order of potency of inorganic blockers on the vasopressin-induced inward current was Gd3+ > La3+ > Cd2+. Vasopressin-induced non-selective cation current was also observed in pertussis toxin-pretreated A7r5 cells but was completely abolished after infusion of the GDP analogue, guanosine 5'-O-[3-thio]diphosphate, from the patch pipette. Furthermore, vasopressin induced a transient outward current, suggesting a Ca(2+)-activated K(+)-current, which overlapped with the nonselective cation conductance.(ABSTRACT TRUNCATED AT 250 WORDS)

Conditioned ("use-dependent") inhibition by phenylalkylamines (PAAs) is a characteristic property of Ltype calcium (Ca 2؉ ) channels. To determine the structural elements of the PAA binding domain we transferred sequence stretches of the pore-forming regions of repeat III and/or IV from the skeletal muscle ␣ 1 subunit (␣ 1S ) to the class A ␣ 1 subunit (␣ 1A ) and expressed these chimeras together with ␤ 1a and ␣ 2 /␦ subunits in Xenopus oocytes. The corresponding barium currents (I Ba ) were tested for PAA sensitivity during trains of depolarizing test pulses (conditioned block). I Ba of oocytes expressing the ␣ 1A subunit were only weakly inhibited by PAAs (less than 10% conditioned block of I Ba during a 100-ms pulse train of 0.1 Hz). Transfer of the transmembrane segment IVS6 from ␣ 1S to ␣ 1A produced an enhancement of PAA sensitivity of the resulting ␣ 1A /␣ 1S chimera comparable to L-type ␣ 1 subunits (about 35% conditioned block of I Ba during a 100-ms pulse train of 0.1 Hz). Our results demonstrate that substitution of 11 amino acids within the segment IVS6 of ␣ 1A with the corresponding residues of ␣ 1S is sufficient to transfer L-type PAA sensitivity into the low sensitive class A Ca 2؉ channel.Voltage-gated Ca 2ϩ channels mediate the depolarization-induced influx of Ca 2ϩ into excitable cells, thereby regulating cellular processes such as muscle contraction, propagation of action potentials, secretion, and gene expression. They are heterooligomeric complexes formed by at least an ␣ 1 , ␤, and ␣ 2 /␦ subunit (1). The ␣ 1 subunit is the pore-forming membrane protein consisting of four homologous repeats (I-IV), each of them composed of six transmembrane segments (S1-S6) (2). Based on different pharmacological and biophysical properties, various types of voltage-dependent Ca 2ϩ channels (T, L, N, P, Q, and R) can be distinguished (3-5). Their sensitivity to Ca (PAAs) (6, 7). In contrast, classes A, B, and E are considered to be DHP-insensitive.To localize Ca 2ϩ antagonist interaction domains within Ltype ␣ 1 subunits we have recently shown (8) that sensitivity for DHP Ca 2ϩ channel blockers and activators can be transferred to class A ␣ 1 subunits (␣ 1A ) by substituting regions close to the channel pore in repeats III and IV (segments IIIS5, IIIS6, IVS6, and the respective S5-S6 linkers) with the corresponding L-type ␣ 1 sequences (from ␣ 1S or ␣ 1C ). The DHP sensitivity was lost after replacement of short sequence stretches within these regions by the ␣ 1A sequence. For example, when segment IIIS5 was replaced by ␣ 1A sequence, DHP sensitivity disappeared. The same effect was observed after replacing the IVS5-IVS6 linker. These results suggest that the DHP molecules interact with multiple amino acid residues located within distant regions of the primary structure.Hockerman et al. (9) recently identified three amino acid residues within segment IVS6 of a L-type Ca 2ϩ channel ␣ 1C subunit (Tyr-1463, Ala-1467, and Ile-1470, numbering according to ␣ 1C-c ) (10) as critical determinants for high affinity ...