Characterization of a potassium(+) 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.