Panulirus interruptus Ih-channel gene PIIH: modification of channel properties by alternative splicing and role in rhythmic activity.
We cloned 10 full-length variants of PIIH, the gene for I(h) from the spiny lobster, Panulirus interruptus, using reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE). This gene shows a significant amount of alternative splicing in the S3-S4 and S4-S5 linkers, in the P-loop and the entire S6 transmembrane domain, in the cyclic nucleotide binding domain (CNBD), and near the 3' end of the gene. Functional expression of seven splice variants in Xenopus oocytes generated slowly activating hyperpolarization-activated inward currents, which were blocked by the I(h) channel blockers CsCl and ZD7288. The different splice variants had markedly varying activation kinetics and voltage dependence of activation. Bath application of 8-Br-cAMP shifted the V(1/2) to more positive potentials and accelerated the activation kinetics in an isoform-specific manner. Two variants containing a segment with an ER-retention motif in the S4-S5 loop did not produce currents in oocytes. Overexpression of one splice variant, PIIH AB(S)-I, in pyloric dilator (PD) neurons in the lobster stomatogastric ganglion produced an average threefold increase in I(h) without evoking a compensatory increase in I(A). The voltage for half-maximal activation of I(h) in PIIH AB(S)-I-expressing PDs was shifted in the depolarizing direction by 9 mV, whereas the slope factor decreased by 3.8 mV. Moreover, its activation kinetics were significantly faster than in control PDs. PIIH AB(S)-I overexpression enhanced PD neuron rhythmic firing in an amplitude-dependent manner above a minimal threshold two- to threefold increase in amplitude.
Ouyang
Qing
Q
Dept. of Neurobiology and Behavior, Cornell University, W159 Seeley G. Mudd Hall, Ithaca, NY 14853. rmh4@cornell.edu).
Goeritz
Marie
M
Harris-Warrick
Ronald M
RM
D017398:Alternative Splicing; D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D054815:Cyclic Nucleotide-Gated Cation Channels; D004558:Electric Stimulation / Q000379:methods; D017952:Ganglia, Invertebrate / Q000166:cytology; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels / Q000235:genetics* / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D008845:Microinjections / Q000379:methods; D009474:Neurons / Q000502:physiology; D009865:Oocytes; D033521:Palinuridae / Q000166:cytology / Q000235:genetics*; D018408:Patch-Clamp Techniques / Q000379:methods; D010507:Periodicity; D015221:Potassium Channels / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000502:physiology; D014981:Xenopus
D054815:Cyclic Nucleotide-Gated Cation Channels; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D015221:Potassium Channels; D020033:Protein Isoforms