Different types of K+ channels play important roles in many aspects of excitability. The isolation of cDNA clones from Drosophila, Aplysia, Xenopus, and mammals points to a large multigene family with several distinct members encoding K+ channels with unique electrophysiological and pharmacological properties. Given the pivotal role K+ channels play in the fine tuning of electrical properties of excitable tissues, we studied the spatial and temporal basis of K+ channel diversity. We report the isolation of two putative K+ channels that define two new subfamilies based upon amino acid sequence similarities with other known K+ channels. Northern blot and in situ hybridization studies revealed differences in the spatial and temporal expression patterns for these two new clones along with mRNAs from other K+ channel subfamilies. Two of the K+ channels studied are predominantly expressed in the brain. One of the "brain-specific" K+ channels is first expressed after about 2 weeks of postnatal cerebellar development and remains at levels about 10-fold higher in the cerebellum than in the rest of the brain.
A novel member of the RCK family of rat brain K+ channels, called RCKZ, has been sequenced and expressed in Xenopus oocytes. The K+ currents were voltage-dependent, activated within 20 ms (at 0 mV), did not inactivate in 5 s, and had a single channel conductance in frog Ringers of 8.2 pS. Compared to other members of the RCK family the pharmacological profile of RCK2 was unique in that the channel was resistant to block (IC,, = 3.3~M) by charybdotoxin [( 1988) Proc. Natl. Acad. Sci. USA 85,3329-33331 but relatively sensitive to %aminopyridine (0.3 mM), tetraethylammonium (I .7 mM), cl-dendrotoxin (25 nM), noxiustoxin (200 nM), and mast cell degranulating peptide (200 nM). Thus, RCK2 is a non-inactivating delayed rectifier K+ channel with interesting pharmacological properties.Potassium channel; cDNA cloning; cDNA expression; Delayed rectifier; Xenopus oocyte 1, INTRODUCTION sitivity to blockers such as 4-aminopyridine, dendrotoxin and mast cell degranulating protein suggests that it Voltage-activated K+ channels are integral membrane may be related to the K' channels which affect neuroproteins which xegulate the transmembrane diffusion of transmitter release in rat brain synaptosomes [I]. M+ ions. Channel activation controls neuronal ex-RCK2, a cDNA encoding a rat brain K+ channel has citability through repolarization of the action potential recently been described by Grupe et al.[ 101. The cDNA and modulation of the frequency of repetitive firing [S].clone reported here encodes a polypeptide which differs From whole-cell and single channel electrophysiological from RCKZ at only one residue and hence will be refermeasurements [25,29], neurons are thought to express red to as RCKZ, also. Our electrophysiological results heterogeneous populations of K+ channels which differ are closely comparable to those obtained by Grupe et al. in their biophysical and pharmacological properties.[IO], except that in our experiments charybdotoxin Until recently the structural basis of K+ channel diversi-(ChTX) was lOOO-fold less potent in blocking K+ surty was not known; however, molecular cloning methods rent than was reported by Grupe et al. [lo]. have now shown that mammalian brain mRNA encodes several distinct voltage-activated K3 channels [2,8,15, 22,24,25]. Expression of cloned rat brain K+ channels 2. MATERIALS AND METHODS provides a new basis for understanding the relationships between primary structure and channel function. Isolation and sequencing of cDNAUsing an oligonucleotide probe encoding a strictly Rat brain cDNA libraries enriched for full-length inserts [7] were screened at low stringency using an oligonucleotide probe encoding non-inactivating delayed rectifier Kf current, Its senbearing an approximately 5 kb insert, gave large K+ currents, and had a DNA sequence which was different from all known K' channels. Electrophysiological recording
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