Characterization of a K+ channel from Limulus photoreceptors: molecular biology and electrophysiology
This thesis describes an investigation of the functional role of potassium (K+) channels in the photoreceptors, of the horseshoe crab, ' Limulus polyphemus'. Molecular cloning combined with electrical recordings can be used to determine the underlying mechanisms of the photoresponse. This thesis describes how the gene encoding a K+ channel from RNA extracted from the ventral eye of the horseshoe crab was cloned using the polymerase chain reaction (PCR), was expressed and characterized in ' Xenopus laevis' oocytes. The unique 533 amino acid sequence making up the 'Limulus' K+ channel (LimK) has a 71% homology to the 'Drosophila ' Shaker K+ channel. LimK expressed in oocytes produced slowly inactivating, K+-dependent, outward currents. The half-maximum voltage of inactivation was -34 mV and the time-constant of inactivation was 1.8s when fit with a single-exponential. LimK activity was insensitive to the externally applied K+-channel blocker tetraethylammonium ion (TEA) and it was abolished by the external application of the K+-channel blocker 4-aminopyridine (4-AP). LimK did not demonstrate the kinetic property of fast-inactivation. Vertebrate channels of the subfamily of voltage-gated K+ channels require the addition of an internal [beta]-subunit in order to undergo fast inactivation. This may explain the absence of fast-inactivation for LimK, since the pharmacological evidence indicates that LimK may act as a fast-inactivating channel within the photoreceptor. 'In vivo' electrophysiological experiments on 'Limulus' photoreceptors have found a fast-inactivating K+ channel that plays a role in shaping the receptor potential. Evidence is presented suggesting that LimK may be this channel.