No abstract

Central neurons have multiple types of voltage-dependent potassium channels, whose activation during action potentials shapes spike width and whose activation and inactivation at subthreshold voltages modulate firing frequency. We characterized the voltage-dependent potassium currents flowing during the action potentials of hippocampal CA3 pyramidal neurons and examined the susceptibility of the underlying channel types to inactivation at subthreshold voltages. Using acutely dissociated neurons that permitted rapid voltage clamp, action potentials recorded previously were used as the command voltage waveform, and individual components of potassium current were identified by pharmacological sensitivity. The overall voltage-dependent potassium current in the neurons could be split into three major components based on pharmacology and kinetics during step voltage pulses: I(D) (fast activating, slowly inactivating, and sensitive to 4-aminopyridine at 30 microm), I(A) (fast activating, fast inactivating, and sensitive to 4-aminopyridine at 3 mm), and I(K) (slowly activating, noninactivating, and sensitive to external TEA at 3-25 mm). The potassium current during the action potential was composed of approximately equal contributions of I(D) and I(A), with a negligible contribution of I(K). I(D) and I(A) had nearly identical trajectories of activation and deactivation during the action potential. Both I(A) and I(D) showed steady-state inactivation at subthreshold voltages, but maximal inactivation at such voltages was incomplete for both currents. Because of the major contribution of both I(D) and I(A) to spike repolarization, it is likely that modulation or partial inactivation at subthreshold voltages of either current can influence spike timing with minimal effect on spike width.

Five novel peptidyl inhibitors of

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 ...

The transmembrane segment IIIS5 of the L-type calcium channel ␣ 1 subunit participates in the formation of the 1,4-dihydropyridine (DHP) interaction domain (Grabner, M., Wang, Z., Hering, S., Striessnig, J., and Glossmann, H. (1996) Neuron 16, 207-218). We applied mutational analysis to identify amino acid residues within this segment that contribute to DHP sensitivity. DHP agonist and antagonist modulation of Ba 2؉ inward currents was assessed after coexpression of chimeric and mutant calcium channel ␣ 1 subunits with ␣ 2 ␦ and ␤ 1a subunits in Xenopus oocytes. Whereas DHP antagonists required Thr-1066, DHP agonist modulation crucially depended on the additional presence of Gln-1070 (numbering according to ␣ 1C-a ), which also further increased the sensitivity to DHP antagonists. Asp-955, which is found at the corresponding position in the calcium channel ␣ 1S subunit from carp skeletal muscle, displayed functional similarity to Gln-1070 with respect to DHP interaction. We conclude that these residues (Thr-1066 plus Gln-1070 or Asp-955), which are located in close vicinity on the same side of the putative ␣-helix of transmembrane segment IIIS5, form a crucial DHP binding motif.Voltage-dependent calcium channels rapidly and selectively mediate calcium entry into excitable cells. According to their electrophysiological and pharmacological properties, N-, L-, T-, P-, Q-, and R-type channels are distinguished (for review, see Refs. 1-4). L-type channel function is modulated by 1,4-dihydropyridine (DHP) 1 calcium agonists and antagonists. The latter are used in the treatment of cardiovascular disorders, such as hypertension and ischemic heart disease (5).To fully understand the molecular mechanism of channel modulation by these drugs, the identification of amino acid residues that are involved in DHP binding to L-type calcium channels is required. Photoaffinity labeling combined with antibody mapping (6), as well as mutational analysis (7-10), identified segments IIIS6 and IVS6 and the S5-S6 linkers in repeats III and IV of the ␣ 1 subunits to form the DHP interaction domain. We recently demonstrated that introducing only as much as 9.4% L-type sequence into the DHP-insensitive class A (BI-2) calcium channel ␣ 1 subunit is sufficient to transfer drug sensitivity (8). A significant new finding of this study was that segment IIIS5 is critically involved in formation of the DHP interaction domain.Here we employed site-directed mutagenesis and heterologous expression in Xenopus oocytes to examine the impact of individual amino acid residues within segment IIIS5 on DHP action. We found that Thr-1066 and Gln-1070 of ␣ 1C-a differentially contribute to DHP agonist and antagonist effects. These residues are located in close vicinity on the same side in a putative ␣-helix of transmembrane segment IIIS5. Since the additional sequence stretches of the DHP binding pocket are formed by putatively pore-lining segments (11), our data suggest that segment IIIS5 must also be located close to or within the pore of L-type calcium chan...