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TRESK (TWIK-related spinal cord K(+) channel) is the most recently identified member of the two-pore-domain potassium channel (K(2P)) family, the molecular source of background potassium currents. Human TRESK channels are not affected by external acidification. However, the mouse orthologue displays moderate pH dependence isolated to a single histidine residue adjacent to the GYG selectivity filter. In the human protein, sequence substitution of tyrosine by histidine at this critical position generated a mutant that displays almost identical proton sensitivity compared with mouse TRESK. In contrast to human TRESK, which is specifically located in spinal cord, we detected mouse TRESK (mTRESK) mRNA in several epithelial and neuronal tissues including lung, liver, kidney, brain and spinal cord. As revealed by endpoint and quantitative RT-PCR, mTRESK channels are mainly expressed in dorsal root ganglia (DRG) and on the transcript level represent the most important background potassium channel in this tissue. DRG neurones of all sizes were labelled by in situ hybridizations with TRESK-specific probes. In DRG neurones of TRESK[G339R] functional knock-out (KO) mice the standing outward current IK(so) was significantly reduced compared with TRESK wild-type (WT) littermates. Different responses to K(2P) channel regulators such as bupivacaine, extracellular protons and quinidine corroborated the finding that approximately 20% of IK(so) is carried by TRESK channels. Unexpectedly, we found no difference in resting membrane potential between DRG neurones of TRESK[WT] and TRESK[G339R] functional KO mice. However, in current-clamp recordings we observed significant changes in action potential duration and amplitude of after-hyperpolarization. Most strikingly, cellular excitability of DRG neurones from functional KO mice was significantly augmented as revealed by reduced rheobase current to elicit action potentials.

Voltage-gated potassium channels, Kv1.1, Kv1.2 and Kv1.6, were identified as PCR products from mRNA prepared from nodose ganglia. Immunocytochemical studies demonstrated expression of the proteins in all neurons from ganglia of neonatal animals (postnatal days 0-3) and in 85-90 % of the neurons from older animals (postnatal days 21-60). In voltage clamp studies, alpha-dendrotoxin (alpha-DTX), a toxin with high specificity for these members of the Kv1 family, was used to examine their contribution to K(+) currents of the sensory neurons. alpha-DTX blocked current in both A- and C-type neurons. The current had characteristics of a delayed rectifier with activation positive to -50 mV and little inactivation during 250 ms pulses. In current-clamp experiments alpha-DTX, used to eliminate the current, had no effect on resting membrane potential and only small effects on the amplitude and duration of the action potential of A- and C-type neurons. However, there were prominent effects on excitability. alpha-DTX lowered the threshold for initiation of discharge in response to depolarizing current steps, reduced spike after-hyperpolarization and increased the frequency/pattern of discharge of A- and C-type neurons at membrane potentials above threshold. Model simulations were consistent with these experimental results and demonstrated how the other major K(+) currents function in response to the loss of the alpha-DTX-sensitive current to effect these changes in action potential wave shape and discharge.

1. Proteins of the Kv4 or Shal-related subfamily are key components of transient K+ channels (A channels) operating at subthreshold values of the membrane potential. We have cloned and characterized a new mammalian Kv4 or Shal-related cDNA (Kv4.3) that predicts a protein with strong sequence conservation with the other known members of this subfamily. 2. Injection of Kv4.3 transcripts into Xenopus oocytes generates an A type K+ current, with small but physiologically significant differences from the currents expressed by Kv4.2 and Kv4.1 mRNAs. Kv4.3 currents can be modified to resemble native A currents by coinjection with a low molecular weight mRNA fraction from rat brain which does not express detectable currents on its own. Particularly striking is a 7-to-10-fold increase in the rate of recovery from inactivation, a 5- to 10-fold increase in current magnitude and a 3- to 4-fold increase in sensitivity to 4-amino pyridine (4-AP). 3. In situ hybridization histochemistry was used to compare the expression of the three known Kv4 genes. Kv4.2 and Kv4.3 (but not Kv4.1) are abundant in the adult rat brain, with each displaying a specific, but sometimes overlapping pattern of expression. Moreover, a reciprocal gradient of expression of Kv4.2 and Kv4.3 transcripts is seen in some brain areas, such as in the pyramidal cell layers of the hippocampus and the granule cell layer of the cerebellum. Therefore Kv4 proteins may form heteromultimeric channels of distinct subunit composition in different neurons. Moreover, the results suggest that neurons such as pyramidal cells in the hippocampus and granule cells in the cerebellum represent heterogeneous cell populations in terms of their ISA, and hence in their firing patterns. Kv4.2 and Kv4.3 also display complementary expression in the heart, with Kv4.3 being more abundant in atria and Kv4.2 in ventricle. The existence of multiple Kv4 proteins forming channels of variable subunit combinations helps explain the diversity of ISA channels in neurons.

1. The blocking action of 4-aminopyridine (4-AP) on a delayed rectifier Kv1.2 K+ channel expressed in oocytes was investigated at room temperature (22 degrees C) and physiological temperature (34 degrees C) using the double-electrode voltage clamp and patch clamp techniques. 2. At room temperature, 4-AP (100 microM) inhibition occurred only after activation of current. The rate of onset of block was dependent upon the length of time current was activated by a depolarizing step. Similarly, removal of block required current activation. The degree of steady-state block by 4-AP was not reduced by increasingly more depolarized step potentials. The degree of steady-state block also did not change over the duration of a 1 s step. 3. When channels were nearly fully inactivated, 4-AP produced no additional block of a subsequent depolarizing step, suggesting that 4-AP did not bind when channels were in the inactivated state. In single channel experiments, 4-AP decreased the mean open time in a dose-dependent manner but did not alter the single-channel current amplitude. 4. At 34 degrees C the I-V relationship and inactivation curve shifted to more negative potentials. Increasing the temperature to 34 degrees C did not alter the degree of block by 4-AP, although the rate of onset of block was greatly enhanced. 5. Results suggest that 4-AP binds to the open state of the Kv1.2 channel and is trapped when the channel closes. 4-AP cannot bind when the channel is closed or inactivated prior to the addition of the drug. C-type inactivation and 4-AP binding to the channel are mutually exclusive. A model for the proposed mechanism of action of 4-AP on the Kv1.2 channel is proposed based on experimental data.