Cumulative inactivation and the pore domain in the Kv1 channels

Abstract

AKv1.1a is an Aplysia Kv1 channel close to a mammalian Kv1.4. Both channels show a prominent frequency-dependent cumulative inactivation. The cumulative inactivation of AKv1.1a but not of rKv1.4 was enhanced by the patch excision. To gain structural information about the phenomenon, we examined chimeras of AKv1.1a and rKv1.4. Chimeras with the AKv1.1a pore domain displayed enhanced cumulative inactivation after patch excision. In the pore domain, eight amino acids are different between the two channels. We, therefore, constructed eight mutants of AKv1.1a (A378E, D379P, Q380T, K384Q, R406K, G409T, W411G, L414I) based on the sequence differences. All the mutants showed a similar macroscopic current decay, suggesting that N-type inactivation was not affected. The patch excision failed to enhance the cumulative inactivation in A378E, D379P, G409T, and L414I. P/C-type inactivation in N-terminal deletion mutants (Delta N) became slower in A378E, D379P, G409T and L414I. Internal application of the N-terminal peptide from the ShB channel induces a frequency-dependent block of Delta N-AKv1.1a. By contrast, the block was not frequency dependent in Delta N-A378E, Delta N-D379P and Delta N-G409T. The results suggest that the positions 378, 379, 409, and perhaps 414 in the AKv1.1a backbone are involved in the stability of P/C-type inactivation and the cumulative inactivation of Kv1 channels.

Molecular cloning and functional characterization of the Aplysia FMRFamide-gated Na+ channel

“…Electrophysiological recording and data acquisition were essentially as described previously [36]. An oocyte was placed at the bottom of an experimental chamber (.100 ll) and continuously perfused with ND96 (.1 ml/min).…”

Stability of N-type inactivation and the coupling between N-type and C-type inactivation in theAplysiaKv1 channel

“…The functional property of N-type inactivation of AKv1 as well as its molecular mechanism are rather well characterized [14, 16, 51, 52, 53, 54, 60]. In the present study, we first reexamined the steady state activation and inactivation of AKv1, I8Q and ΔN and estimated the half activation/inactivation voltage (V half ) and the slope factor (k).…”

“…If the effect of external Zn 2+ on the inactivation of AKv1 is mostly dependent on C- type inactivation, Zn 2+ should have little effect on the inactivation of the mutant channels which have impaired C-type mechanism. We previously examined several mutants around the pore turret region of AKv1, and found that some mutants show deteriorated C-type inactivation with intact N-type mechanism [60]. Among the previously described mutants, we checked two mutants, A378E and D379P, to see the effect of Zn 2+ on their inactivation.…”

“…An Aplysia Kv1 channel (AKv1) [52] was used in the present study. The plasmids containing the wild-type AKv1 (pSPAK01) and the amino-terminal deletion mutant of AKv1 (ΔN, pSPAK01ΔN, the 2nd–61th amino acids were removed) as well as the plasmids containing mutants (pSPAK01-A378E, pSPAK01-D379P, pSPAK01ΔN-A378E) were described previously [15, 60]. I8Q mutant of AKv1 (pSPAK01-I8Q) as well as H382Q mutants (pSPAK01-H382Q, pSPAK01ΔN-H382Q) were made by Quick Change protocol (Agilent Technologies, Santa Clara, CA, USA).…”

“…Membrane currents of oocytes were measured by two-electrode voltage clamp by using OC-725C (Warner Instruments LLC., Hamden, CT, USA) as described previously [60]. An oocyte was placed in a recording chamber ( ∼ 100 µ l), and continuously perfused with ND96 by gravity ( ∼ 2 ml/min).…”