Mutations in the S4 domain of a pacemaker channel alter its voltage dependence

In an attempt to study the functional role of the positively charged amino acids present in the S4 segment of hyperpolarization-activated cyclic nucle otide-gated cation (HCN) channels, rye have introduced single and sequentia l amino acid replacements throughout this domain in the mouse type 2 HCN ch annel (mHCN2), Sequential neutralization of the first three positively char ged amino acids resulted in cumulative shifts of the midpoint voltage activ ation constant towards more hyperpolarizing potentials, The contribution of each amino acid substitution was approximately -20 mV, Amino acid replacem ents to neutralize either the first (K291Q) or fourth (R300Q) positively ch arged amino acid resulted in the same shift (about -20 mV) towards more hyp erpolarized potentials, Replacing the first positively charged amino acid w ith the negatively charged glutamic acid (K291E) produced a shift of approx imately -50 mV in the same direction, None of the above amino acid substitu tions had any measurable effect on the time course of channel activation. T his suggests that the S4 domain of HCN channels critically controls the vol tage dependence of channel opening but is not involved in regulating activa tion kinetics. No channel activity was detected in mutants with neutralizat ion of the last sis positively charged amino acids from the S4 domain, sugg esting that these amino acids cannot be altered without impairing channel f unction, (C) 2000 Federation of European Biochemical Societies. Published b y Elsevier Science B.V. All rights reserved.