Kvbeta1.1 associates with Kvalpha1.4 in Chinese hamster ovary cells and pigeon type II vestibular hair cells and enhances the amplitude, inactivation and negatively shifts the steady-state inactivation range.
Although A-type potassium currents are found in type II hair cells in the inner ear of most species, the molecular mechanisms for activation and inactivation of the A-type potassium current (I(A)) remain unknown. In frog semicircular canal hair cells, for example, there appear to be two classes of currents having either fast or slow inactivation [Norris CH, Ricci AJ, Housley GD, Guth PS (1992) The inactivating potassium currents of hair cells isolated from the crista ampullaris of the frog. J Neurophysiol 68:1642-1653; Russo G, Calzi D, Martini M, Rossi ML, Fesce R, Prigioni I (2007) Potassium currents in the hair cells of vestibular epithelium: position-dependent expression of two types of A channels. Eur J Neurosci 25:695-704]. It has been suggested that somehow the "ball and chain" mechanism (NH(3) (N) terminus motif) is modified by alternative splicing to account for the two classes of inactivation. To examine other possibilities, we cloned alpha and beta subunits that comprise the A-type potassium channel complex in adult and embryonic pigeon brain, cochlea and labyrinth. By sequence homology, we concluded that the subunits present were Kvalpha1.4 and Kvbeta1.1. The sequence of the open reading frame for Kvalpha1.4 contained the N-terminus, pore and COOH (C) terminus motifs for N-and C-type inactivation. The sequence for Kvbeta1.1 displayed amino acids consistent with assembly and association with Kvalpha1.4 alpha subunits. Kvalpha1.4 and Kvbeta1.1 were transfected either singly or in combination into Chinese hamster ovary (CHO) cells. These cells and native hair cells from the pigeon utricle were patch clamped and the inactivation properties of the A-type current were studied. In the native hair cells, the A-type current was identified by its pharmacological (4-aminopyridine (4-AP); IC(50)=11 microM) and voltage dependent inactivation properties. A comparison of the mean time constants from best-fitted single exponential and sum of two exponential equations to the ionic current inactivation revealed the following. In CHO cells when Kvalpha1.4 was expressed alone, the mean time constant (tau(1)=107 ms+/-19, N=32) was significantly (P<0.001) longer and the mean peak amplitude (2.28 nA+/-0.39, N=32) was smaller than when Kvalpha1.4 and Kvbeta1.1 were expressed in CHO cells. Moreover, the co-transfection of Kvalpha1.4 and Kvbeta1.1 into CHO cells caused a shift in the steady state inactivation curve parameter Vo 30 mV in the hyperpolarized direction relative to CHO cells expressing only Kvalpha1.4. Similarly, Kvalpha1.4-transfected CHO cells produced longer time constants and smaller amplitudes than those found for native utricular hair cells. These data lead us to conclude that while the amino acid motifs are present in Kvalpha1.4 and Kvbeta1.1 to suggest N-and C-type inactivation, co-assembly and association of Kvalpha1.4 and Kvbeta1.1 may also produce changes in the time dependent inactivation properties of vestibular hair cells.
Correia
M J
MJ
Department of Otolaryngology, Room 7.102 Blocker Medical Research Building (Route 1063), UTMB, Galveston, TX 77555-1063, USA. mjcorrei@utmb.edu
Weng
T
T
Prusak
D
D
Wood
T G
TG
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D003001:Cloning, Molecular; D010856:Columbidae; D006224:Cricetinae; D003412:Cricetulus; D018069:Hair Cells, Vestibular / Q000166:cytology / Q000378:metabolism*; D015640:Ion Channel Gating / Q000235:genetics; D040542:Mechanotransduction, Cellular / Q000235:genetics; D008564:Membrane Potentials / Q000235:genetics; D009924:Organ Culture Techniques; D018408:Patch-Clamp Techniques; D024642:Potassium Channels, Voltage-Gated / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012444:Saccule and Utricle / Q000166:cytology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014162:Transfection; D014722:Vestibule, Labyrinth / Q000166:cytology / Q000378:metabolism*
D024642:Potassium Channels, Voltage-Gated; D021122:Protein Subunits