Determinants of activation kinetics in mammalian hyperpolarization-activated cation channels.

1. The structural basis for the different activation kinetics of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels was investigated with the whole-cell patch clamp technique by using HCN1, HCN4, chimeric channels and mutants in a mammalian expression system (COS-7). 2. The activation time constant of HCN4 was about 40-fold longer than that of HCN1 when compared at -100 mV. 3. In chimeras between HCN1 and HCN4, the region of the S1 transmembrane domain and the exoplasmic S1-S2 linker markedly affected the activation kinetics. The cytoplasmic region between S6 and the cyclic nucleotide-binding domain (CNBD) also significantly affected the activation kinetics. 4. The S1 domain and S1-S2 linker of HCN1 differ from those of HCN4 at eight amino acid residues, and each single point mutation of them changed the activation kinetics less than 2-fold. However, the effects of those mutations were additive and the substitution of the whole S1 and S1-S2 region of HCN1 by that of HCN4 resulted in a 10- to 20-fold slowing. 5. The results indicate that S1 and S1-S2, and S6-CNBD are the crucial components for the activation gating of HCN channels.

Inhibition of the human intermediate conductance Ca(2+)-activated K(+) channel, hIK1, by volatile anesthetics.

Ca(2+)-activated K(+) channels (K(Ca)) regulate a wide variety of cellular functions by coupling intracellular Ca(2+) concentration to membrane potential. There are three major groups of K(Ca) classified by their unit conductances: large (BK), intermediate (IK), and small (SK) conductance of channels. BK channel is gated by combined influences of Ca(2+) and voltage, while IK and SK channels are gated solely by Ca(2+). Volatile anesthetics inhibit BK channel activity by interfering with the Ca(2+) gating mechanism. However, the effects of anesthetics on IK and SK channels are unknown. Using cloned IK and SK channels, hIK1 and hSK1-3, respectively, we found that the currents of hIK1 were inhibited rapidly and reversibly by volatile anesthetics, whereas those of SK channels were not affected. The IC(50) values of the volatile anesthetics, halothane, sevoflurane, enflurane, and isoflurane for hIK1 inhibition were 0.69, 0.42, 1.01 and 1.03 mM, respectively, and were in the clinically used concentration range. In contrast to BK channel, halothane inhibition of hIK1 currents was independent of Ca(2+) concentration, suggesting that Ca(2+) gating mechanism is not involved. These results demonstrate that volatile anesthetics, such as halothane, enflurane, isoflurane, and sevoflurane, affect BK, IK, and SK channels in distinct ways.

Molecular characterization of the hyperpolarization-activated cation channel in rabbit heart sinoatrial node.

We cloned a cDNA (HAC4) that encodes the hyperpolarization-activated cation channel (If or Ih) by screening a rabbit sinoatrial (SA) node cDNA library using a fragment of rat brain If cDNA. HAC4 is composed of 1150 amino acid residues, and its cytoplasmic N- and C-terminal regions are longer than those of HAC1-3. The transmembrane region of HAC4 was most homologous to partially cloned mouse If BCNG-3 (96%), whereas the C-terminal region of HAC4 showed low homology to all HAC family members so far cloned. Northern blotting revealed that HAC4 mRNA was the most highly expressed in the SA node among the rabbit cardiac tissues examined. The electrophysiological properties of HAC4 were examined using the whole cell patch-clamp technique. In COS-7 cells transfected with HAC4 cDNA, hyperpolarizing voltage steps activated slowly developing inward currents. The half-maximal activation was obtained at -87.2 +/- 2.8 mV under control conditions and at -64.4 +/- 2.6 mV in the presence of intracellular 0.3 mM cAMP. The reversal potential was -34.2 +/- 0.9 mV in 140 mM Na+o and 5 mM K+o versus 10 mM Na+i and 145 mM K+i. These results indicate that HAC4 forms If in rabbit heart SA node.

A human intermediate conductance calcium-activated potassium channel.

An intermediate conductance calcium-activated potassium channel, hIK1, was cloned from human pancreas. The predicted amino acid sequence is related to, but distinct from, the small conductance calcium-activated potassium channel subfamily, which is approximately 50% conserved. hIK1 mRNA was detected in peripheral tissues but not in brain. Expression of hIK1 in Xenopus oocytes gave rise to inwardly rectifying potassium currents, which were activated by submicromolar concentrations of intracellular calcium (K0.5 = 0.3 microM). Although the K0.5 for calcium was similar to that of small conductance calcium-activated potassium channels, the slope factor derived from the Hill equation was significantly reduced (1.7 vs. 3. 5). Single-channel current amplitudes reflected the macroscopic inward rectification and revealed a conductance level of 39 pS in the inward direction. hIK1 currents were reversibly blocked by charybdotoxin (Ki = 2.5 nM) and clotrimazole (Ki = 24.8 nM) but were minimally affected by apamin (100 nM), iberiotoxin (50 nM), or ketoconazole (10 microM). These biophysical and pharmacological properties are consistent with native intermediate conductance calcium-activated potassium channels, including the erythrocyte Gardos channel.

Determinants of apamin and d-tubocurarine block in SK potassium channels.

Small conductance calcium-activated potassium channels show a distinct pharmacology. Some, but not all, are blocked by the peptide toxin apamin, and apamin-sensitive channels are also blocked by d-tubocurarine. Cloned SK channels (small conductance calcium-activated potassium channel) recapitulate these properties. We have investigated the structural basis for these differences and found that two amino acid residues on either side of the deep pore are the primary determinants of sensitivity to apamin and differential block by d-tubocurarine. Therefore, the pharmacology of SK channels compared with other potassium channels correlates with structural differences in the outer pore region. However, introduction of a tyrosine residue in the position analogous to that which determines sensitivity to external tetraethylammonium for voltage-gated potassium channels endows SK channels with an equivalent tetraethylammonium sensitivity, indicating that the outer vestibules of the pores are similar. The pharmacology of channels formed in oocytes coinjected with SK1 and SK2 mRNAs, or with SK1-SK2 dimer mRNA, show that SK subunits may form heteromeric channels.