Cloning, expression, and distribution of functionally distinct Ca(2+)-activated K+ channel isoforms from human brain.

We have cloned and expressed nine Ca(2+)-activated K+ channel isoforms from human brain. The open reading frames encode proteins ranging from 1154 to 1195 amino acids, and all possess significant identity with the slowpoke gene products in Drosophila and mouse. All isoforms are generated by alternative RNA splicing of a single gene on chromosome 10 at band q22.3 (hslo). RNA splicing occurs at four sites located in the carboxy-terminal portion of the protein and gives rise to at least nine ion channel constructs (hbr1-hbr9). hslo mRNA is expressed abundantly in human brain, and individual isoforms show unique expression patterns. Expression of hslo mRNA in Xenopus oocytes produces robust voltage and Ca(2+)-activated K+ currents. Splice variants differ significantly in their Ca2+ sensitivity, suggesting a broad functional role for these channels in the regulation of neuronal excitability.

Redox modulation of hslo Ca2+-activated K+ channels.

The modulation of ion channel proteins by cellular redox potential has emerged recently as a significant determinant of channel function. We have investigated the influence of sulfhydryl redox reagents on human brain Ca2+-activated K+ channels (hslo) expressed in both human embryonic kidney 293 cells and Xenopus oocytes using macropatch and single-channel analysis. Intracellular application of the reducing agent dithiothreitol (DTT): (1) shifts the voltage of half-maximal channel activation (V0.5) approximately 18 mV to more negative potentials without affecting the maximal conductance or the slope of the voltage dependence; (2) slows by approximately 10-fold a time-dependent right-shift in V0.5 values ("run-down"); (3) speeds macroscopic current activation kinetics by approximately 33%; and (4) increases the single-channel open probability without affecting the unitary conductance. In contrast to DTT treatment, oxidation with hydrogen peroxide shifts macropatch V0.5 values to more positive potentials, increases the rate of channel run-down, and decreases the single-channel open probability. KCa channels cloned from Drosophila differ from hslo channels in that they show very little run-down and are not modulated by the addition of DTT. These data indicate that hslo Ca2+-activated K+ channels may be modulated by changes in the cellular redox potential as well as by the transmembrane voltage and the cytoplasmic Ca2+ concentration.

Distinct effects of Ca2+ and voltage on the activation and deactivation of cloned Ca(2+)-activated K+ channels.

1. Cloned large-conductance Ca(2+)-activated K+ channels from Drosophila (dslo) and human (hslo) were expressed in Xenopus oocytes. The effects of Ca2+ and voltage on these channels were compared by analysing both macroscopic currents and single-channel transitions. 2. The activation kinetics of dslo Ca(2+)-activated K+ channels are strongly influenced by the intracellular Ca2+ concentration, but are only minimally affected by membrane voltage. Current activation kinetics increase more than 60-fold in response to Ca2+ concentration increases in the range 0.56-405 microM, but increase less than 2-fold by voltage changes from -60 to +80 mV. 3. The activation kinetics of hslo channels are similarly influenced by increases in Ca2+ concentration; however, these kinetics are also increased 5- to 10-fold by voltage changes from -60 to +80 mV. 4. The deactivation kinetics of both dslo and hslo channels are also more Ca2+ sensitive than voltage sensitive. Increasing concentrations of Ca2+ slow deactivation kinetics more than 40-fold, while changes in the membrane voltage cause less than 2-fold changes. 5. Ca2+ increases the activation kinetics by altering first latency distributions. Increasing the Ca2+ concentration from 0.56 to 2.4 microM causes a 20-fold decrease in the mean time to first channel opening. 6. Both Ca2+ and voltage have large effects on regulating the steady-state open probability of these ion channels. Plots relating open probability (Po) to membrane voltage show a voltage dependence of 16.5 mV per e-fold change in Po for dslo and 12.3 mV per e-fold change in Po for hslo. At any given voltage the Ca2+ sensitivity of dslo is lower than that for hslo. The Hill coefficient for Ca2+ activation is 1.9 +/- 0.15, indicating that the binding of at least two Ca2+ ions is required to maximally activate both dslo and hslo channels. 7. The gating kinetics of both dslo and hslo channels can be well described by three open and five closed states. Changing the free Ca2+ concentration alters the time constants for the three longest closed states, without affecting any of the open states. Changing the membrane voltage alters the same three closed states, as well as the longest of the three open states. The two shortest occupancy open and closed time constants underlying these states are largely independent of voltage and Ca2+. 8. To account for these data, we propose that Ca2+ binding to the closed channel is the slow rate-limiting step in the activation pathway and, conversely, that Ca2+ unbinding is the slow rate-limiting step in the deactivation pathway. Hence, Ca2+ appears to bind to the closed channel and allows it to undergo a number of slow conformational changes that bring the channel to a state from which it can quickly open upon depolarization. These data imply that while both Ca2+ and voltage can alter the steady-state open probability of these channels, only Ca2+ has large effects on altering non-steady-state parameters and thus is the intracellular signal that predominantly modulates the rate of channel activation and deactivation.

Activation of metabotropic glutamate receptors differentially affects two classes of hippocampal interneurons and potentiates excitatory synaptic transmission.

Based on responses to metabotropic glutamate receptor (mGluR) activation, we have characterized two distinct classes of interneuron in stratum (st.) oriens of the CA1 region of hippocampus. One type of interneuron was strongly excited by 1S,3R-aminocyclopentane dicarboxylic acid (ACPD), responding with a large inward current accompanied by increased baseline noise and prominent current oscillations. A second interneuron population responded with a modest inward current with no changes in baseline noise. These two classes of responses persisted in the presence of tetrodotoxin and antagonists of ionotropic glutamate and GABA receptors, suggesting that the inward currents result from mGluRs on the interneurons themselves. The two physiologically defined cell types correspond to two distinct morphological cell types in st. oriens/alveus, distinguished by very different patterns of local axonal connections. Large oscillatory inward current responses were recorded predominantly from an interneuron type whose axons heavily innervated st. lacunosum. The more modest inward current response was generally found in interneurons whose axons innervated the somata and proximal dendrites of CA1 pyramidal neurons. These differences in physiology and local circuitry imply that activation of mGluRs in st. oriens will cause very strong excitation of interneurons synapsing in st. lacunosum, and weaker excitation of interneurons innervating pyramidal cells at the soma and proximal dendrites. These data suggest that each interneuron population has a specific role in hippocampal function, and that mGluR activation will affect the local circuit differently for each interneuron type. Metabotropic GluR activation also markedly enhanced the amplitudes of the evoked and spontaneous EPSCs received by all interneurons in the region, independent of changes in the postsynaptic holding current and with no change in the kinetics of the EPSC. In contrast to the enhancement of evoked and spontaneous EPSCs, miniature EPSCs recorded in the presence of tetrodotoxin were not increased. These data suggest that ACPD acts at a presynaptic site to potentiate the EPSC. Taken together, these results highlight an important modulatory role for metabotropic receptors located at sites both pre- and postsynaptic to CA1 st. oriens interneurons.